Deep learning-based variant classifier

ABSTRACT

The technology disclosed directly operates on sequencing data and derives its own feature filters. It processes a plurality of aligned reads that span a target base position. It combines elegant encoding of the reads with a lightweight analysis to produce good recall and precision using lightweight hardware. For instance, one million training examples of target base variant sites with 50 to 100 reads each can be trained on a single GPU card in less than 10 hours with good recall and precision. A single GPU card is desirable because it a computer with a single GPU is inexpensive, almost universally within reach for users looking at genetic data. It is readily available on could-based platforms.

PRIORITY APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/247,487, entitled “Deep Learning-Based Variant Classifier,” whichclaims priority to or the benefit of U.S. Provisional Patent ApplicationNo. 62/617,552, entitled “DEEP LEARNING-BASED VARIANT CLASSIFIER,” filedon Jan. 15, 2018, (Atty. Docket No. ILLM 1005-1/IP-1663-PRV). Thepriority application is hereby incorporated by reference for allpurposes.

INCORPORATIONS

The following are incorporated by reference for all purposes as if fullyset forth herein:

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FIELD OF THE TECHNOLOGY DISCLOSED

The technology disclosed relates to artificial intelligence typecomputers and digital data processing systems and corresponding dataprocessing methods and products for emulation of intelligence (i.e.,knowledge based systems, reasoning systems, and knowledge acquisitionsystems); and including systems for reasoning with uncertainty (e.g.,fuzzy logic systems), adaptive systems, machine learning systems, andartificial neural networks. In particular, the technology disclosedrelates to using deep learning and convolutional neural networks (CNNs)for analyzing ordered data.

BACKGROUND

The subject matter discussed in this section should not be assumed to beprior art merely as a result of its mention in this section. Similarly,a problem mentioned in this section or associated with the subjectmatter provided as background should not be assumed to have beenpreviously recognized in the prior art. The subject matter in thissection merely represents different approaches, which in and ofthemselves can also correspond to implementations of the claimedtechnology.

Accurate identification of variant in genetic sequences has manyimportant impacts and has garnered significant attention. The latesteffort to apply Google's Inception engine to variant calling isinteresting, but extremely resource intensive. A more efficient approachis needed.

Next-generation sequencing has made large amounts of sequenced dataavailable for variant classification. Sequenced data are highlycorrelated and have complex interdependencies, which has hindered theapplication of traditional classifiers like support vector machine tothe variant classification task. Advanced classifiers that are capableof extracting high-level features from sequenced data are thus desired.

Deep neural networks are a type of artificial neural networks that usemultiple nonlinear and complex transforming layers to successively modelhigh-level features and provide feedback via backpropagation. Deepneural networks have evolved with the availability of large trainingdatasets, the power of parallel and distributed computing, andsophisticated training algorithms. Deep neural networks have facilitatedmajor advances in numerous domains such as computer vision, speechrecognition, and natural language processing.

Convolutional neural networks and recurrent neural networks arecomponents of deep neural networks. Convolutional neural networks havesucceeded particularly in image recognition with an architecture thatcomprises convolution layers, nonlinear layers, and pooling layers.Recurrent neural networks are designed to utilize sequential informationof input data with cyclic connections among building blocks likeperceptrons, long short-term memory units, and gated recurrent units. Inaddition, many other emergent deep neural networks have been proposedfor limited contexts, such as deep spatio-temporal neural networks,multi-dimensional recurrent neural networks, and convolutionalauto-encoders.

The goal of training deep neural networks is optimization of the weightparameters in each layer, which gradually combines simpler features intocomplex features so that the most suitable hierarchical representationscan be learned from data. A single cycle of the optimization process isorganized as follows. First, given a training dataset, the forward passsequentially computes the output in each layer and propagates thefunction signals forward through the network. In the final output layer,an objective loss function measures error between the inferenced outputsand the given labels. To minimize the training error, the backward passuses the chain rule to backpropagate error signals and compute gradientswith respect to all weights throughout the neural network. Finally, theweight parameters are updated using optimization algorithms based onstochastic gradient descent. Whereas batch gradient descent performsparameter updates for each complete dataset, stochastic gradient descentprovides stochastic approximations by performing the updates for eachsmall set of data examples. Several optimization algorithms stem fromstochastic gradient descent. For example, the Adagrad and Adam trainingalgorithms perform stochastic gradient descent while adaptivelymodifying learning rates based on update frequency and moments of thegradients for each parameter, respectively.

Another core element in the training of deep neural networks isregularization, which refers to strategies intended to avoid overfittingand thus achieve good generalization performance. For example, weightdecay adds a penalty term to the objective loss function so that weightparameters converge to smaller absolute values. Dropout randomly removeshidden units from neural networks during training and can be consideredan ensemble of possible subnetworks. To enhance the capabilities ofdropout, a new activation function, maxout, and a variant of dropout forrecurrent neural networks called rnnDrop have been proposed.Furthermore, batch normalization provides a new regularization methodthrough normalization of scalar features for each activation within amini-batch and learning each mean and variance as parameters.

Given that sequenced data are multi- and high-dimensional, deep neuralnetworks have great promise for bioinformatics research because of theirbroad applicability and enhanced prediction power. Convolutional neuralnetworks have been adapted to solve sequence-based problems in genomicssuch as motif discovery, pathogenic variant identification, and geneexpression inference. A hallmark of convolutional neural networks is theuse of convolution filters. Unlike traditional classification approachesthat are based on elaborately-designed and manually-crafted features,convolution filters perform adaptive learning of features, analogous toa process of mapping raw input data to the informative representation ofknowledge. In this sense, the convolution filters serve as a series ofmotif scanners, since a set of such filters is capable of recognizingrelevant patterns in the input and updating themselves during thetraining procedure. Recurrent neural networks can capture long-rangedependencies in sequential data of varying lengths, such as protein orDNA sequences.

Therefore, an opportunity arises to use deep neural networks for variantclassification.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. The color drawings also may be available in PAIRvia the Supplemental Content tab. In the drawings, like referencecharacters generally refer to like parts throughout the different views.Also, the drawings are not necessarily to scale, with an emphasisinstead generally being placed upon illustrating the principles of thetechnology disclosed. In the following description, variousimplementations of the technology disclosed are described with referenceto the following drawings, in which:

FIG. 1A shows one implementation of variant calling by a trained variantclassifier disclosed herein. The trained variant classifier includes aconvolutional neural network (CNN).

FIG. 1B illustrates one implementation of training the variantclassifier of FIG. 1A using labeled training data comprising candidatevariants.

FIG. 1C depicts one implementation of input and output modules ofconvolutional neural network processing of the variant classifier ofFIG. 1A.

FIG. 2 is one implementation of an array of input features that is fedto the convolutional neural network of the variant classifier of FIG.1A.

FIG. 3A illustrates one implementation of architecture of theconvolutional neural network of the variant classifier of FIG. 1A.

FIG. 3B illustrates another implementation of the architecture of theconvolutional neural network of the variant classifier of FIG. 1A.

FIG. 3C illustrates yet another implementation of the architecture ofthe convolutional neural network of the variant classifier of FIG. 1A.

FIG. 3D illustrates yet another implementation of the architecture ofthe convolutional neural network of the variant classifier of FIG. 1A.

FIG. 4A depicts a fully-connected (FC) network.

FIG. 4B illustrates one implementation of architecture of thefully-connected neural network of the variant classifier that takes asinput only empirical variant score (EVS) features. This architecturedoes not use any convolutions.

FIG. 5 shows one example of precision-recall curves that comparesingle-base polymorphism (SNP) classification performance by theconvolutional neural network of the variant classifier and by a baselineStrelka™ model called empirical variant score (EVS) model.

FIG. 6 shows another example of precision-recall curves that compare SNPclassification performance by the convolutional neural network of thevariant classifier and by the EVS model.

FIG. 7 depicts one example of precision-recall curves that compare indelclassification performance by the convolutional neural network of thevariant classifier and by the EVS model.

FIG. 8 illustrates convergence curves of the variant classifier duringtraining and validation.

FIG. 9 illustrates convergence curves of the fully-connected neuralnetwork of the variant classifier during training and testing(inference).

FIG. 10 uses precision-recall curves to compare SNP classificationperformance of (i) the fully-connected neural network of the variantclassifier trained on EVS features of the EVS model version 2.8.2, (ii)the fully-connected neural network of the variant classifier trained onEVS features of the EVS model version 2.9.2, (iii) the EVS model version2.8.2, and (iv) the EVS model version 2.9.2.

FIG. 11 uses precision-recall curves to compare indel classificationperformance of (i) the fully-connected neural network of the variantclassifier trained on EVS features of the EVS model version 2.8.2, (ii)the fully-connected neural network of the variant classifier trained onEVS features of the EVS model version 2.9.2, (iii) the EVS model version2.8.2, and (iv) the EVS model version 2.9.2.

FIG. 12 is a simplified block diagram of a computer system that can beused to implement the variant classifier.

DETAILED DESCRIPTION

The following discussion is presented to enable any person skilled inthe art to make and use the technology disclosed, and is provided in thecontext of a particular application and its requirements. Variousmodifications to the disclosed implementations will be readily apparentto those skilled in the art, and the general principles defined hereinmay be applied to other implementations and applications withoutdeparting from the spirit and scope of the technology disclosed. Thus,the technology disclosed is not intended to be limited to theimplementations shown, but is to be accorded the widest scope consistentwith the principles and features disclosed herein.

Introduction

The technology disclosed directly operates on DNA sequencing data andderives its own feature filters. It processes plurality of aligned reads(e.g., read depth ranging from 10 to 500) that span a target baseposition. It combines elegant encoding of the reads with a lightweightanalysis to produce good recall and precision using lightweighthardware. For instance, one million training examples of target basevariant sites with 50 to 100 reads each can be trained on a single GPUcard in less than 10 hours with good recall and precision. A single GPUcard is desirable because it a computer with a single GPU isinexpensive, almost universally within reach for users looking atgenetic data. It is readily available on could-based platforms.

Elegant encoding combines the following data for reads centered on atarget base, flanked on each side by 110 bases or more. Of course, few,if any reads will span the 221 base sequence, so most reads will havenull bases on one or both ends of the read sequence. The data encodedfor each base in a read sequence includes the individual read, acorresponding reference base from a reference read, a base call accuracyscore from reading the base, a deoxyribonucleic acid (abbreviated DNA)strandedness of reading the base, an insertion count of insertionchanges adjoining the base, and deletion flag to indicate that alignmentdetermined that the read had a deletion at the individual read site.

In this encoding, insertions and deletions are handled differently.Between the positions of any two reads there can be an arbitrary numberof insertions. The count of his number of insertions is used torepresent an arbitrary number between reference positions. The calls ofthe inserted bases are not used, because misalignment among reads wouldresult. Deletions take place at a particular position that can beflagged. If there are multiple deletions between two individual reads,after alignment, multiple deletion flags can be set at the deletionsites. A deleted base should not be assigned an ACGT code, as noneapplies.

This is a simple encoding system, not involving translation into a colorspace or adaption for processing by an image handling engine such asInception. Simplicity contributes to fast training.

When more computing resources are available, sequences longer than 221base positions can be used. As platforms evolve to produce longer readsequence, advantages of using more flanking bases are expected to becomeapparent.

The per-read data above can be supplemented by per-variantcharacterization data generated by a legacy system, during training andoptionally during operation. There are many rule-based, hand-craftedsystems that characterize variants at specific positions. One or moreinputs, per-variant, can be used as inputs after processing the multiplereads through convolutional layers. The late added, per-variant inputshortens training. This is expected, because the accuracy of legacysystems is already high, estimated in excess of 90 percent.

The lightweight analysis structure also contributes to fast training. Insome embodiments, five convolutional layers for processing the per-readdata, followed by a two layer fully connected structure that acceptsinput from the convolutional output and from the per-variant data hasproven to be a lightweight and accurate network structure. Success alsohas been achieved with seven and eight convolutional layers, so two toeight layers work and more layers could be used.

In more detail, the first convolutional layer accepts the listedencoding in a 221 (base) by 100 (reads) by 12 (attributes, with one-hotencoding of ACGT reads). The center base is taken as the targetposition. A number of randomly initialized or previously trained filtersare applied. In one design, 32 convolution filters are used at a layer.Multi-dimensional filters tend to collapse rows.

With a million training and verification samples available, seventraining epochs has given good results. The number of training epochsshould be limited to avoid overfitting. Limiting the number of epochscan be combined with dropouts to avoid overfitting.

Terminology

All literature and similar material cited in this application,including, but not limited to, patents, patent applications, articles,books, treatises, and web pages, regardless of the format of suchliterature and similar materials, are expressly incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

As used herein, the following terms have the meanings indicated.

A base refers to a nucleotide base or nucleotide, A (adenine), C(cytosine), T (thymine), or G (guanine). This application uses “base(s)”and “nucleotide(s)” interchangeably.

The term “chromosome” refers to the heredity-bearing gene carrier of aliving cell, which is derived from chromatin strands comprising DNA andprotein components (especially histones). The conventionalinternationally recognized individual human genome chromosome numberingsystem is employed herein.

The term “site” refers to a unique position (e.g., chromosome ID,chromosome position and orientation) on a reference genome. In someimplementations, a site may be a residue, a sequence tag, or a segment'sposition on a sequence. The term “locus” may be used to refer to thespecific location of a nucleic acid sequence or polymorphism on areference chromosome.

The term “sample” herein refers to a sample, typically derived from abiological fluid, cell, tissue, organ, or organism containing a nucleicacid or a mixture of nucleic acids containing at least one nucleic acidsequence that is to be sequenced and/or phased. Such samples include,but are not limited to sputum/oral fluid, amniotic fluid, blood, a bloodfraction, fine needle biopsy samples (e.g., surgical biopsy, fine needlebiopsy, etc.), urine, peritoneal fluid, pleural fluid, tissue explant,organ culture and any other tissue or cell preparation, or fraction orderivative thereof or isolated therefrom. Although the sample is oftentaken from a human subject (e.g., patient), samples can be taken fromany organism having chromosomes, including, but not limited to dogs,cats, horses, goats, sheep, cattle, pigs, etc. The sample may be useddirectly as obtained from the biological source or following apretreatment to modify the character of the sample. For example, suchpretreatment may include preparing plasma from blood, diluting viscousfluids and so forth. Methods of pretreatment may also involve, but arenot limited to, filtration, precipitation, dilution, distillation,mixing, centrifugation, freezing, lyophilization, concentration,amplification, nucleic acid fragmentation, inactivation of interferingcomponents, the addition of reagents, lysing, etc.

The term “sequence” includes or represents a strand of nucleotidescoupled to each other. The nucleotides may be based on DNA or RNA. Itshould be understood that one sequence may include multiplesub-sequences. For example, a single sequence (e.g., of a PCR amplicon)may have 350 nucleotides. The sample read may include multiplesub-sequences within these 350 nucleotides. For instance, the sampleread may include first and second flanking subsequences having, forexample, 20-50 nucleotides. The first and second flanking sub-sequencesmay be located on either side of a repetitive segment having acorresponding sub-sequence (e.g., 40-100 nucleotides). Each of theflanking sub-sequences may include (or include portions of) a primersub-sequence (e.g., 10-30 nucleotides). For ease of reading, the term“sub-sequence” will be referred to as “sequence,” but it is understoodthat two sequences are not necessarily separate from each other on acommon strand. To differentiate the various sequences described herein,the sequences may be given different labels (e.g., target sequence,primer sequence, flanking sequence, reference sequence, and the like).Other terms, such as “allele,” may be given different labels todifferentiate between like objects. The application uses “read(s)” and“sequence read(s)” interchangeably.

The term “paired-end sequencing” refers to sequencing methods thatsequence both ends of a target fragment. Paired-end sequencing mayfacilitate detection of genomic rearrangements and repetitive segments,as well as gene fusions and novel transcripts. Methodology forpaired-end sequencing are described in PCT publication WO07010252, PCTapplication Serial No. PCTGB2007/003798 and US patent applicationpublication US 2009/0088327, each of which is incorporated by referenceherein. In one example, a series of operations may be performed asfollows; (a) generate clusters of nucleic acids; (b) linearize thenucleic acids; (c) hybridize a first sequencing primer and carry outrepeated cycles of extension, scanning and deblocking, as set forthabove; (d) “invert” the target nucleic acids on the flow cell surface bysynthesizing a complimentary copy; (e) linearize the resynthesizedstrand; and (f) hybridize a second sequencing primer and carry outrepeated cycles of extension, scanning and deblocking, as set forthabove. The inversion operation can be carried out be delivering reagentsas set forth above for a single cycle of bridge amplification.

The term “reference genome” or “reference sequence” refers to anyparticular known genome sequence, whether partial or complete, of anyorganism which may be used to reference identified sequences from asubject. For example, a reference genome used for human subjects as wellas many other organisms is found at the National Center forBiotechnology Information at ncbi.nlm.nih.gov. A “genome” refers to thecomplete genetic information of an organism or virus, expressed innucleic acid sequences. A genome includes both the genes and thenoncoding sequences of the DNA. The reference sequence may be largerthan the reads that are aligned to it. For example, it may be at leastabout 100 times larger, or at least about 1000 times larger, or at leastabout 10,000 times larger, or at least about 105 times larger, or atleast about 106 times larger, or at least about 107 times larger. In oneexample, the reference genome sequence is that of a full length humangenome. In another example, the reference genome sequence is limited toa specific human chromosome such as chromosome 13. In someimplementations, a reference chromosome is a chromosome sequence fromhuman genome version hg19. Such sequences may be referred to aschromosome reference sequences, although the term reference genome isintended to cover such sequences. Other examples of reference sequencesinclude genomes of other species, as well as chromosomes,sub-chromosomal regions (such as strands), etc., of any species. Invarious implementations, the reference genome is a consensus sequence orother combination derived from multiple individuals. However, in certainapplications, the reference sequence may be taken from a particularindividual. In other implementations, the “genome” also covers so-called“graph genomes”, which use a particular storage format andrepresentation of the genome sequence. In one implementation, graphgenomes store data in a linear file. In another implementation, thegraph genomes refer to a representation where alternative sequences(e.g., different copies of a chromosome with small differences) arestored as different paths in a graph. Additional information regardinggraph genome implementations can be found inhttps://www.biorxiv.org/content/biorxiv/early/2018/03/20/194530.full.pdf,the content of which is hereby incorporated herein by reference in itsentirety.

The term “read” refer to a collection of sequence data that describes afragment of a nucleotide sample or reference. The term “read” may referto a sample read and/or a reference read. Typically, though notnecessarily, a read represents a short sequence of contiguous base pairsin the sample or reference. The read may be represented symbolically bythe base pair sequence (in ATCG) of the sample or reference fragment. Itmay be stored in a memory device and processed as appropriate todetermine whether the read matches a reference sequence or meets othercriteria. A read may be obtained directly from a sequencing apparatus orindirectly from stored sequence information concerning the sample. Insome cases, a read is a DNA sequence of sufficient length (e.g., atleast about 25 bp) that can be used to identify a larger sequence orregion, e.g., that can be aligned and specifically assigned to achromosome or genomic region or gene.

Next-generation sequencing methods include, for example, sequencing bysynthesis technology (Illumina), pyrosequencing (454), ion semiconductortechnology (Ion Torrent sequencing), single-molecule real-timesequencing (Pacific Biosciences) and sequencing by ligation (SOLiDsequencing). Depending on the sequencing methods, the length of eachread may vary from about 30 bp to more than 10,000 bp. For example, theDNA sequencing method using SOLiD sequencer generates nucleic acid readsof about 50 bp. For another example, Ion Torrent Sequencing generatesnucleic acid reads of up to 400 bp and 454 pyrosequencing generatesnucleic acid reads of about 700 bp. For yet another example,single-molecule real-time sequencing methods may generate reads of10,000 bp to 15,000 bp. Therefore, in certain implementations, thenucleic acid sequence reads have a length of 30-100 bp, 50-200 bp, or50-400 bp.

The terms “sample read”, “sample sequence” or “sample fragment” refer tosequence data for a genomic sequence of interest from a sample. Forexample, the sample read comprises sequence data from a PCR ampliconhaving a forward and reverse primer sequence. The sequence data can beobtained from any select sequence methodology. The sample read can be,for example, from a sequencing-by-synthesis (SBS) reaction, asequencing-by-ligation reaction, or any other suitable sequencingmethodology for which it is desired to determine the length and/oridentity of a repetitive element. The sample read can be a consensus(e.g., averaged or weighted) sequence derived from multiple samplereads. In certain implementations, providing a reference sequencecomprises identifying a locus-of-interest based upon the primer sequenceof the PCR amplicon.

The term “raw fragment” refers to sequence data for a portion of agenomic sequence of interest that at least partially overlaps adesignated position or secondary position of interest within a sampleread or sample fragment. Non-limiting examples of raw fragments includea duplex stitched fragment, a simplex stitched fragment, a duplexun-stitched fragment and a simplex un-stitched fragment. The term “raw”is used to indicate that the raw fragment includes sequence data havingsome relation to the sequence data in a sample read, regardless ofwhether the raw fragment exhibits a supporting variant that correspondsto and authenticates or confirms a potential variant in a sample read.The term “raw fragment” does not indicate that the fragment necessarilyincludes a supporting variant that validates a variant call in a sampleread. For example, when a sample read is determined by a variant callapplication to exhibit a first variant, the variant call application maydetermine that one or more raw fragments lack a corresponding type of“supporting” variant that may otherwise be expected to occur given thevariant in the sample read.

The terms “mapping”, “aligned,” “alignment,” or “aligning” refer to theprocess of comparing a read or tag to a reference sequence and therebydetermining whether the reference sequence contains the read sequence.If the reference sequence contains the read, the read may be mapped tothe reference sequence or, in certain implementations, to a particularlocation in the reference sequence. In some cases, alignment simplytells whether or not a read is a member of a particular referencesequence (i.e., whether the read is present or absent in the referencesequence). For example, the alignment of a read to the referencesequence for human chromosome 13 will tell whether the read is presentin the reference sequence for chromosome 13. A tool that provides thisinformation may be called a set membership tester. In some cases, analignment additionally indicates a location in the reference sequencewhere the read or tag maps to. For example, if the reference sequence isthe whole human genome sequence, an alignment may indicate that a readis present on chromosome 13, and may further indicate that the read ison a particular strand and/or site of chromosome 13.

The term “indel” refers to the insertion and/or the deletion of bases inthe DNA of an organism. A micro-indel represents an indel that resultsin a net change of 1 to 50 nucleotides. In coding regions of the genome,unless the length of an indel is a multiple of 3, it will produce aframeshift mutation. Indels can be contrasted with point mutations. Anindel inserts and deletes nucleotides from a sequence, while a pointmutation is a form of substitution that replaces one of the nucleotideswithout changing the overall number in the DNA. Indels can also becontrasted with a Tandem Base Mutation (TBM), which may be defined assubstitution at adjacent nucleotides (primarily substitutions at twoadjacent nucleotides, but substitutions at three adjacent nucleotideshave been observed.

The term “variant” refers to a nucleic acid sequence that is differentfrom a nucleic acid reference. Typical nucleic acid sequence variantincludes without limitation single nucleotide polymorphism (SNP), shortdeletion and insertion polymorphisms (Indel), copy number variation(CNV), microsatellite markers or short tandem repeats and structuralvariation. Somatic variant calling is the effort to identify variantspresent at low frequency in the DNA sample. Somatic variant calling isof interest in the context of cancer treatment. Cancer is caused by anaccumulation of mutations in DNA. A DNA sample from a tumor is generallyheterogeneous, including some normal cells, some cells at an early stageof cancer progression (with fewer mutations), and some late-stage cells(with more mutations). Because of this heterogeneity, when sequencing atumor (e.g., from an FFPE sample), somatic mutations will often appearat a low frequency. For example, a SNV might be seen in only 10% of thereads covering a given base. A variant that is to be classified assomatic or germline by the variant classifier is also referred to hereinas the “variant under test”.

The term “noise” refers to a mistaken variant call resulting from one ormore errors in the sequencing process and/or in the variant callapplication.

The term “variant frequency” represents the relative frequency of anallele (variant of a gene) at a particular locus in a population,expressed as a fraction or percentage. For example, the fraction orpercentage may be the fraction of all chromosomes in the population thatcarry that allele. By way of example, sample variant frequencyrepresents the relative frequency of an allele/variant at a particularlocus/position along a genomic sequence of interest over a “population”corresponding to the number of reads and/or samples obtained for thegenomic sequence of interest from an individual. As another example, abaseline variant frequency represents the relative frequency of anallele/variant at a particular locus/position along one or more baselinegenomic sequences where the “population” corresponding to the number ofreads and/or samples obtained for the one or more baseline genomicsequences from a population of normal individuals.

The term “variant allele frequency (VAF)” refers to the percentage ofsequenced reads observed matching the variant divided by the overallcoverage at the target position. VAF is a measure of the proportion ofsequenced reads carrying the variant.

The terms “position”, “designated position”, and “locus” refer to alocation or coordinate of one or more nucleotides within a sequence ofnucleotides. The terms “position”, “designated position”, and “locus”also refer to a location or coordinate of one or more base pairs in asequence of nucleotides.

The term “haplotype” refers to a combination of alleles at adjacentsites on a chromosome that are inherited together. A haplotype may beone locus, several loci, or an entire chromosome depending on the numberof recombination events that have occurred between a given set of loci,if any occurred.

The term “threshold” herein refers to a numeric or non-numeric valuethat is used as a cutoff to characterize a sample, a nucleic acid, orportion thereof (e.g., a read). A threshold may be varied based uponempirical analysis. The threshold may be compared to a measured orcalculated value to determine whether the source giving rise to suchvalue suggests should be classified in a particular manner. Thresholdvalues can be identified empirically or analytically. The choice of athreshold is dependent on the level of confidence that the user wishesto have to make the classification. The threshold may be chosen for aparticular purpose (e.g., to balance sensitivity and selectivity). Asused herein, the term “threshold” indicates a point at which a course ofanalysis may be changed and/or a point at which an action may betriggered. A threshold is not required to be a predetermined number.Instead, the threshold may be, for instance, a function that is based ona plurality of factors. The threshold may be adaptive to thecircumstances. Moreover, a threshold may indicate an upper limit, alower limit, or a range between limits.

In some implementations, a metric or score that is based on sequencingdata may be compared to the threshold. As used herein, the terms“metric” or “score” may include values or results that were determinedfrom the sequencing data or may include functions that are based on thevalues or results that were determined from the sequencing data. Like athreshold, the metric or score may be adaptive to the circumstances. Forinstance, the metric or score may be a normalized value. As an exampleof a score or metric, one or more implementations may use count scoreswhen analyzing the data. A count score may be based on number of samplereads. The sample reads may have undergone one or more filtering stagessuch that the sample reads have at least one common characteristic orquality. For example, each of the sample reads that are used todetermine a count score may have been aligned with a reference sequenceor may be assigned as a potential allele. The number of sample readshaving a common characteristic may be counted to determine a read count.Count scores may be based on the read count. In some implementations,the count score may be a value that is equal to the read count. In otherimplementations, the count score may be based on the read count andother information. For example, a count score may be based on the readcount for a particular allele of a genetic locus and a total number ofreads for the genetic locus. In some implementations, the count scoremay be based on the read count and previously-obtained data for thegenetic locus. In some implementations, the count scores may benormalized scores between predetermined values. The count score may alsobe a function of read counts from other loci of a sample or a functionof read counts from other samples that were concurrently run with thesample-of-interest. For instance, the count score may be a function ofthe read count of a particular allele and the read counts of other lociin the sample and/or the read counts from other samples. As one example,the read counts from other loci and/or the read counts from othersamples may be used to normalize the count score for the particularallele.

The terms “coverage” or “fragment coverage” refer to a count or othermeasure of a number of sample reads for the same fragment of a sequence.A read count may represent a count of the number of reads that cover acorresponding fragment. Alternatively, the coverage may be determined bymultiplying the read count by a designated factor that is based onhistorical knowledge, knowledge of the sample, knowledge of the locus,etc.

The term “read depth” (conventionally a number followed by “x”) refersto the number of sequenced reads with overlapping alignment at thetarget position. This is often expressed as an average or percentageexceeding a cutoff over a set of intervals (such as exons, genes, orpanels). For example, a clinical report might say that a panel averagecoverage is 1,105× with 98% of targeted bases covered >100×.

The terms “base call quality score” or “Q score” refer to a PHRED-scaledprobability ranging from 0-50 inversely proportional to the probabilitythat a single sequenced base is correct. For example, a T base call withQ of 20 is considered likely correct with a probability of 99.99%. Anybase call with Q<20 should be considered low quality, and any variantidentified where a substantial proportion of sequenced reads supportingthe variant are of low quality should be considered potentially falsepositive.

The terms “variant reads” or “variant read number” refer to the numberof sequenced reads supporting the presence of the variant.

Regarding “strandedness” (or DNA strandedness), the genetic message inDNA can be represented as a string of the letters A, G, C, and T. Forexample, 5′-AGGACA-3′. Often, the sequence is written in the directionshown here, i.e., with the 5′ end to the left and the 3′ end to theright. DNA may sometimes occur as single-stranded molecule (as incertain viruses), but normally we find DNA as a double-stranded unit. Ithas a double helical structure with two antiparallel strands. In thiscase, the word “antiparallel” means that the two strands run inparallel, but have opposite polarity. The double-stranded DNA is heldtogether by pairing between bases and the pairing is always such thatadenine (A) pairs with thymine (T) and cytosine (C) pairs with guanine(G). This pairing is referred to as complementarity, and one strand ofDNA is said to be the complement of the other. The double-stranded DNAmay thus be represented as two strings, like this: 5′-AGGACA-3′ and3′-TCCTGT-5′. Note that the two strands have opposite polarity.Accordingly, the strandedness of the two DNA strands can be referred toas the reference strand and its complement, forward and reverse strands,top and bottom strands, sense and antisense strands, or Watson and Crickstrands.

The reads alignment (also called reads mapping) is the process offiguring out where in the genome a sequence is from. Once the alignmentis performed, the “mapping quality” or the “mapping quality score(MAPQ)” of a given read quantifies the probability that its position onthe genome is correct. The mapping quality is encoded in the phred scalewhere P is the probability that the alignment is not correct. Theprobability is calculated as: P=10^((−MAQ/10)), where MAPQ is themapping quality. For example, a mapping quality of 40=10 to the power of−4, meaning that there is a 0.01% chance that the read was alignedincorrectly. The mapping quality is therefore associated with severalalignment factors, such as the base quality of the read, the complexityof the reference genome, and the paired-end information. Regarding thefirst, if the base quality of the read is low, it means that theobserved sequence might be wrong and thus its alignment is wrong.Regarding the second, the mappability refers to the complexity of thegenome. Repeated regions are more difficult to map and reads falling inthese regions usually get low mapping quality. In this context, the MAPQreflects the fact that the reads are not uniquely aligned and that theirreal origin cannot be determined. Regarding the third, in case ofpaired-end sequencing data, concordant pairs are more likely to be wellaligned. The higher is the mapping quality, the better is the alignment.A read aligned with a good mapping quality usually means that the readsequence was good and was aligned with few mismatches in a highmappability region. The MAPQ value can be used as a quality control ofthe alignment results. The proportion of reads aligned with an MAPQhigher than 20 is usually for downstream analysis.

Sequencing Process

Implementations set forth herein may be applicable to analyzing nucleicacid sequences to identify sequence variations. Implementations may beused to analyze potential variants/alleles of a genetic position/locusand determine a genotype of the genetic locus or, in other words,provide a genotype call for the locus. By way of example, nucleic acidsequences may be analyzed in accordance with the methods and systemsdescribed in US Patent Application Publication No. 2016/0085910 and USPatent Application Publication No. 2013/0296175, the complete subjectmatter of which are expressly incorporated by reference herein in theirentirety.

In one implementation, a sequencing process includes receiving a samplethat includes or is suspected of including nucleic acids, such as DNA.The sample may be from a known or unknown source, such as an animal(e.g., human), plant, bacteria, or fungus. The sample may be takendirectly from the source. For instance, blood or saliva may be takendirectly from an individual. Alternatively, the sample may not beobtained directly from the source. Then, one or more processors directthe system to prepare the sample for sequencing. The preparation mayinclude removing extraneous material and/or isolating certain material(e.g., DNA). The biological sample may be prepared to include featuresfor a particular assay. For example, the biological sample may beprepared for sequencing-by-synthesis (SBS). In certain implementations,the preparing may include amplification of certain regions of a genome.For instance, the preparing may include amplifying predetermined geneticloci that are known to include STRs and/or SNPs. The genetic loci may beamplified using predetermined primer sequences.

Next, the one or more processors direct the system to sequence thesample. The sequencing may be performed through a variety of knownsequencing protocols. In particular implementations, the sequencingincludes SBS. In SBS, a plurality of fluorescently-labeled nucleotidesare used to sequence a plurality of clusters of amplified DNA (possiblymillions of clusters) present on the surface of an optical substrate(e.g., a surface that at least partially defines a channel in a flowcell). The flow cells may contain nucleic acid samples for sequencingwhere the flow cells are placed within the appropriate flow cellholders.

The nucleic acids can be prepared such that they comprise a known primersequence that is adjacent to an unknown target sequence. To initiate thefirst SBS sequencing cycle, one or more differently labeled nucleotides,and DNA polymerase, etc., can be flowed into/through the flow cell by afluid flow subsystem. Either a single type of nucleotide can be added ata time, or the nucleotides used in the sequencing procedure can bespecially designed to possess a reversible termination property, thusallowing each cycle of the sequencing reaction to occur simultaneouslyin the presence of several types of labeled nucleotides (e.g., A, C, T,G). The nucleotides can include detectable label moieties such asfluorophores. Where the four nucleotides are mixed together, thepolymerase is able to select the correct base to incorporate and eachsequence is extended by a single base. Non-incorporated nucleotides canbe washed away by flowing a wash solution through the flow cell. One ormore lasers may excite the nucleic acids and induce fluorescence. Thefluorescence emitted from the nucleic acids is based upon thefluorophores of the incorporated base, and different fluorophores mayemit different wavelengths of emission light. A deblocking reagent canbe added to the flow cell to remove reversible terminator groups fromthe DNA strands that were extended and detected. The deblocking reagentcan then be washed away by flowing a wash solution through the flowcell. The flow cell is then ready for a further cycle of sequencingstarting with introduction of a labeled nucleotide as set forth above.The fluidic and detection operations can be repeated several times tocomplete a sequencing run. Example sequencing methods are described, forexample, in Bentley et al., Nature 456:53-59 (2008), InternationalPublication No. WO 04/018497; U.S. Pat. No. 7,057,026; InternationalPublication No. WO 91/06678; International Publication No. WO 07/123744;U.S. Pat. Nos. 7,329,492; 7,211,414; 7,315,019; 7,405,281, and U.S.Patent Application Publication No. 2008/0108082, each of which isincorporated herein by reference.

In some implementations, nucleic acids can be attached to a surface andamplified prior to or during sequencing. For example, amplification canbe carried out using bridge amplification to form nucleic acid clusterson a surface. Useful bridge amplification methods are described, forexample, in U.S. Pat. No. 5,641,658; U.S. Patent Application PublicationNo. 2002/0055100; U.S. Pat. No. 7,115,400; U.S. Patent ApplicationPublication No. 2004/0096853; U.S. Patent Application Publication No.2004/0002090; U.S. Patent Application Publication No. 2007/0128624; andU.S. Patent Application Publication No. 2008/0009420, each of which isincorporated herein by reference in its entirety. Another useful methodfor amplifying nucleic acids on a surface is rolling circleamplification (RCA), for example, as described in Lizardi et al., Nat.Genet. 19:225-232 (1998) and U.S. Patent Application Publication No.2007/0099208 A1, each of which is incorporated herein by reference.

One example SBS protocol exploits modified nucleotides having removable3′ blocks, for example, as described in International Publication No. WO04/018497, U.S. Patent Application Publication No. 2007/0166705A1, andU.S. Pat. No. 7,057,026, each of which is incorporated herein byreference. For example, repeated cycles of SBS reagents can be deliveredto a flow cell having target nucleic acids attached thereto, forexample, as a result of the bridge amplification protocol. The nucleicacid clusters can be converted to single stranded form using alinearization solution. The linearization solution can contain, forexample, a restriction endonuclease capable of cleaving one strand ofeach cluster. Other methods of cleavage can be used as an alternative torestriction enzymes or nicking enzymes, including inter alia chemicalcleavage (e.g., cleavage of a diol linkage with periodate), cleavage ofabasic sites by cleavage with endonuclease (for example ‘USER’, assupplied by NEB, Ipswich, Mass., USA, part number M5505S), by exposureto heat or alkali, cleavage of ribonucleotides incorporated intoamplification products otherwise comprised of deoxyribonucleotides,photochemical cleavage or cleavage of a peptide linker. After thelinearization operation a sequencing primer can be delivered to the flowcell under conditions for hybridization of the sequencing primer to thetarget nucleic acids that are to be sequenced.

A flow cell can then be contacted with an SBS extension reagent havingmodified nucleotides with removable 3′ blocks and fluorescent labelsunder conditions to extend a primer hybridized to each target nucleicacid by a single nucleotide addition. Only a single nucleotide is addedto each primer because once the modified nucleotide has beenincorporated into the growing polynucleotide chain complementary to theregion of the template being sequenced there is no free 3′-OH groupavailable to direct further sequence extension and therefore thepolymerase cannot add further nucleotides. The SBS extension reagent canbe removed and replaced with scan reagent containing components thatprotect the sample under excitation with radiation. Example componentsfor scan reagent are described in U.S. Patent Application PublicationNo. 2008/0280773 A1 and U.S. patent application Ser. No. 13/018,255,each of which is incorporated herein by reference. The extended nucleicacids can then be fluorescently detected in the presence of scanreagent. Once the fluorescence has been detected, the 3′ block may beremoved using a deblock reagent that is appropriate to the blockinggroup used. Example deblock reagents that are useful for respectiveblocking groups are described in WO004018497, US 2007/0166705A1 and U.S.Pat. No. 7,057,026, each of which is incorporated herein by reference.The deblock reagent can be washed away leaving target nucleic acidshybridized to extended primers having 3′-OH groups that are nowcompetent for addition of a further nucleotide. Accordingly the cyclesof adding extension reagent, scan reagent, and deblock reagent, withoptional washes between one or more of the operations, can be repeateduntil a desired sequence is obtained. The above cycles can be carriedout using a single extension reagent delivery operation per cycle wheneach of the modified nucleotides has a different label attached thereto,known to correspond to the particular base. The different labelsfacilitate discrimination between the nucleotides added during eachincorporation operation. Alternatively, each cycle can include separateoperations of extension reagent delivery followed by separate operationsof scan reagent delivery and detection, in which case two or more of thenucleotides can have the same label and can be distinguished based onthe known order of delivery.

Although the sequencing operation has been discussed above with respectto a particular SBS protocol, it will be understood that other protocolsfor sequencing any of a variety of other molecular analyses can becarried out as desired.

Then, the one or more processors of the system receive the sequencingdata for subsequent analysis. The sequencing data may be formatted invarious manners, such as in a .BAM file. The sequencing data mayinclude, for example, a number of sample reads. The sequencing data mayinclude a plurality of sample reads that have corresponding samplesequences of the nucleotides. Although only one sample read isdiscussed, it should be understood that the sequencing data may include,for example, hundreds, thousands, hundreds of thousands, or millions ofsample reads. Different sample reads may have different numbers ofnucleotides. For example, a sample read may range between 10 nucleotidesto about 500 nucleotides or more. The sample reads may span the entiregenome of the source(s). As one example, the sample reads are directedtoward predetermined genetic loci, such as those genetic loci havingsuspected STRs or suspected SNPs.

Each sample read may include a sequence of nucleotides, which may bereferred to as a sample sequence, sample fragment or a target sequence.The sample sequence may include, for example, primer sequences, flankingsequences, and a target sequence. The number of nucleotides within thesample sequence may include 30, 40, 50, 60, 70, 80, 90, 100 or more. Insome implementations, one or more the sample reads (or sample sequences)includes at least 150 nucleotides, 200 nucleotides, 300 nucleotides, 400nucleotides, 500 nucleotides, or more. In some implementations, thesample reads may include more than 1000 nucleotides, 2000 nucleotides,or more. The sample reads (or the sample sequences) may include primersequences at one or both ends.

Next, the one or more processors analyze the sequencing data to obtainpotential variant call(s) and a sample variant frequency of the samplevariant call(s). The operation may also be referred to as a variant callapplication or variant caller. Thus, the variant caller identifies ordetects variants and the variant classifier classifies the detectedvariants as somatic or germline. Alternative variant callers may beutilized in accordance with implementations herein, wherein differentvariant callers may be used based on the type of sequencing operationbeing performed, based on features of the sample that are of interestand the like. One non-limiting example of a variant call application,such as the Pisces™ application by Illumina Inc. (San Diego, CA) hostedat https://github.com/Illumina/Pisces and described in the article Dunn,Tamsen & Berry, Gwenn & Emig-Agius, Dorothea & Jiang, Yu & Iyer, Anita &Udar, Nitin & Stromberg, Michael. (2017). Pisces: An Accurate andVersatile Single Sample Somatic and Germline Variant Caller. 595-595.10.1145/3107411.3108203, the complete subject matter of which isexpressly incorporated herein by reference in its entirety.

Such a variant call application can comprise four sequentially executedmodules:

-   -   (1) Pisces Read Stitcher: Reduces noise by stitching paired        reads in a BAM (read one and read two of the same molecule) into        consensus reads. The output is a stitched BAM.    -   (2) Pisces Variant Caller: Calls small SNVs, insertions and        deletions. Pisces includes a variant-collapsing algorithm to        coalesce variants broken up by read boundaries, basic filtering        algorithms, and a simple Poisson-based variant        confidence-scoring algorithm. The output is a VCF.    -   (3) Pisces Variant Quality Recalibrator (VQR): In the event that        the variant calls overwhelmingly follow a pattern associated        with thermal damage or FFPE deamination, the VQR step will        downgrade the variant Q score of the suspect variant calls. The        output is an adjusted VCF.    -   (4) Pisces Variant Phaser (Scylla): Uses a read-backed greedy        clustering method to assemble small variants into complex        alleles from clonal subpopulations. This allows for the more        accurate determination of functional consequence by downstream        tools. The output is an adjusted VCF.

Additionally or alternatively, the operation may utilize the variantcall application Strelka™ application by Illumina Inc. hosted athttps://github.com/Illumina/strelka and described in the article TSaunders, Christopher & Wong, Wendy & Swamy, Sajani & Becq, Jennifer & JMurray, Lisa & Cheetham, Keira. (2012). Strelka: Accurate somaticsmall-variant calling from sequenced tumor-normal sample pairs.Bioinformatics (Oxford, England). 28. 1811-7.10.1093/bioinformatics/bts271, the complete subject matter of which isexpressly incorporated herein by reference in its entirety. Furthermore,additionally or alternatively, the operation may utilize the variantcall application Strelka2™ application by Illumina Inc. hosted athttps://github.com/Illumina/strelka and described in the article Kim,S., Scheffler, K., Halpern, A. L., Bekritsky, M. A., Noh, E., Milberg,M., Chen, X., Beyter, D., Krusche, P., and Saunders, C. T. (2017).Strelka2: Fast and accurate variant calling for clinical sequencingapplications, the complete subject matter of which is expresslyincorporated herein by reference in its entirety. Moreover, additionallyor alternatively, the operation may utilize a variant annotation/calltool, such as the Nirvana™ application by Illumina Inc. hosted athttps://github.com/Illumina/Nirvana/wiki and described in the articleStromberg, Michael & Roy, Rajat & Lajugie, Julien & Jiang, Yu & Li,Haochen & Margulies, Elliott. (2017). Nirvana: Clinical Grade VariantAnnotator. 596-596. 10.1145/3107411.3108204, the complete subject matterof which is expressly incorporated herein by reference in its entirety.

Such a variant annotation/call tool can apply different algorithmictechniques such as those disclosed in Nirvana:

-   -   a. Identifying all overlapping transcripts with Interval Array:        For functional annotation, we can identify all transcripts        overlapping a variant and an interval tree can be used. However,        since a set of intervals can be static, we were able to further        optimize it to an Interval Array. An interval tree returns all        overlapping transcripts in O(min(n,k lg n)) time, where n is the        number of intervals in the tree and k is the number of        overlapping intervals. In practice, since k is really small        compared to n for most variants, the effective runtime on        interval tree would be O(k lg n). We improved to O(lg n+k) by        creating an interval array where all intervals are stored in a        sorted array so that we only need to find the first overlapping        interval and then enumerate through the remaining (k−1).    -   b. CNVs/SVs (Yu): annotations for Copy Number Variation and        Structural Variants can be provided. Similar to the annotation        of small variants, transcripts overlapping with the SV and also        previously reported structural variants can be annotated in        online databases. Unlike the small variants, not all overlapping        transcripts need be annotated, since too many transcripts will        be overlapped with a large SVs. Instead, all overlapping        transcripts can be annotated that belong to a partial        overlapping gene. Specifically, for these transcripts, the        impacted introns, exons and the consequences caused by the        structural variants can be reported. An option to allow output        all overlapping transcripts is available, but the basic        information for these transcripts can be reported, such as gene        symbol, flag whether it is canonical overlap or partial        overlapped with the transcripts. For each SV/CNV, it is also of        interest to know if these variants have been studied and their        frequencies in different populations. Hence, we reported        overlapping SVs in external databases, such as 1000 genomes, DGV        and ClinGen. To avoid using an arbitrary cutoff to determine        which SV is overlapped, instead all overlapping transcripts can        be used and the reciprocal overlap can be calculated, i.e. the        overlapping length divided by the minimum of the length of these        two SVs.    -   c. Reporting supplementary annotations: Supplementary        annotations are of two types: small and structural variants        (SVs). SVs can be modeled as intervals and use the interval        array discussed above to identify overlapping SVs. Small        variants are modeled as points and matched by position and        (optionally) allele. As such, they are searched using a        binary-search-like algorithm. Since the supplementary annotation        database can be quite large, a much smaller index is created to        map chromosome positions to file locations where the        supplementary annotation resides. The index is a sorted array of        objects (made up of chromosome position and file location) that        can be binary searched using position. To keep the index size        small, multiple positions (up to a certain max count) are        compressed to one object that stores the values for the first        position and only deltas for subsequent positions. Since we use        Binary search, the runtime is O(lg n), where n is the number of        items in the database.    -   d. VEP cache files    -   e. Transcript Database: The Transcript Cache (cache) and        Supplementary database (SAdb) files are serialized dump of data        objects such as transcripts and supplementary annotations. We        use Ensembl VEP cache as our data source for cache. To create        the cache, all transcripts are inserted in an interval array and        the final state of the array is stored in the cache files. Thus,        during annotation, we only need to load a pre-computed interval        array and perform searches on it. Since the cache is loaded up        in memory and searching is very fast (described above), finding        overlapping transcripts is extremely quick in Nirvana (profiled        to less than 1% of total runtime?).    -   f. Supplementary Database: The data sources for SAdb are listed        under supplementary material. The SAdb for small variants is        produced by a k-way merge of all data sources such that each        object in the database (identified by reference name and        position) holds all relevant supplementary annotations. Issues        encountered during parsing data source files have been        documented in detail in Nirvana's home page. To limit memory        usage, only the SA index is loaded up in memory. This index        allows a quick lookup of the file location for a supplementary        annotation. However, since the data has to be fetched from disk,        adding supplementary annotation has been identified as Nirvana's        largest bottleneck (profiled at −30% of total runtime.)    -   g. Consequence and Sequence Ontology: Nirvana's functional        annotation (when provided) follows the Sequence Ontology (SO)        (http://www.sequenceontology.org/) guidelines. On occasions, we        had the opportunity to identify issues in the current SO and        collaborate with the SO team to improve the state of annotation.

Such a variant annotation tool can include pre-processing. For example,Nirvana included a large number of annotations from External datasources, like ExAC, EVS, 1000 Genomes project, dbSNP, ClinVar, Cosmic,DGV and ClinGen. To make full use of these databases, we have tosanitize the information from them. We implemented different strategy todeal with different conflicts that exist from different data sources.For example, in case of multiple dbSNP entries for the same position andalternate allele, we join all ids into a comma separated list of ids; ifthere are multiple entries with different CAF values for the sameallele, we use the first CAF value. For conflicting ExAC and EVSentries, we consider the number of sample counts and the entry withhigher sample count is used. In 1000 Genome Projects, we removed theallele frequency of the conflicting allele. Another issue is inaccurateinformation. We mainly extracted the allele frequencies information from1000 Genome Projects, however, we noticed that for GRCh38, the allelefrequency reported in the info field did not exclude samples withgenotype not available, leading to deflated frequencies for variantswhich are not available for all samples. To guarantee the accuracy ofour annotation, we use all of the individual level genotype to computethe true allele frequencies. As we know, the same variants can havedifferent representations based on different alignments. To make sure wecan accurately report the information for already identified variants,we have to preprocess the variants from different resources to make themhave consistent representation. For all external data sources, wetrimmed alleles to remove duplicated nucleotides in both referenceallele and alternative allele. For ClinVar, we directly parsed the xmlfile we performed a five-prime alignment for all variants, which isoften used in vcf file. Different databases can contain the same set ofinformation. To avoid unnecessary duplicates, we removed some duplicatedinformation. For example, we removed variants in DGV which has datasource as 1000 genome projects, since we already reported these variantsin 1000 genomes with more detailed information.

In accordance with at least some implementations, the variant callapplication provides calls for low frequency variants, germline callingand the like. As non-limiting example, the variant call application mayrun on tumor-only samples and/or tumor-normal paired samples. Thevariant call application may search for single nucleotide variations(SNV), multiple nucleotide variations (MNV), indels and the like. Thevariant call application identifies variants, while filtering formismatches due to sequencing or sample preparation errors. For eachvariant, the variant caller identifies the reference sequence, aposition of the variant, and the potential variant sequence(s) (e.g., Ato C SNV, or AG to A deletion). The variant call application identifiesthe sample sequence (or sample fragment), a reference sequence/fragment,and a variant call as an indication that a variant is present. Thevariant call application may identify raw fragments, and output adesignation of the raw fragments, a count of the number of raw fragmentsthat verify the potential variant call, the position within the rawfragment at which a supporting variant occurred and other relevantinformation. Non-limiting examples of raw fragments include a duplexstitched fragment, a simplex stitched fragment, a duplex un-stitchedfragment and a simplex un-stitched fragment.

The variant call application may output the calls in various formats,such as in a .VCF or .GVCF file. By way of example only, the variantcall application may be included in a MiSeqReporter pipeline (e.g., whenimplemented on the MiSeq® sequencer instrument). Optionally, theapplication may be implemented with various workflows. The analysis mayinclude a single protocol or a combination of protocols that analyze thesample reads in a designated manner to obtain desired information.

Then, the one or more processors perform a validation operation inconnection with the potential variant call. The validation operation maybe based on a quality score, and/or a hierarchy of tiered tests, asexplained hereafter. When the validation operation authenticates orverifies that the potential variant call, the validation operationpasses the variant call information (from the variant call application)to the sample report generator. Alternatively, when the validationoperation invalidates or disqualifies the potential variant call, thevalidation operation passes a corresponding indication (e.g., a negativeindicator, a no call indicator, an in-valid call indicator) to thesample report generator. The validation operation also may pass aconfidence score related to a degree of confidence that the variant callis correct or the in-valid call designation is correct.

Next, the one or more processors generate and store a sample report. Thesample report may include, for example, information regarding aplurality of genetic loci with respect to the sample. For example, foreach genetic locus of a predetermined set of genetic loci, the samplereport may at least one of provide a genotype call; indicate that agenotype call cannot be made; provide a confidence score on a certaintyof the genotype call; or indicate potential problems with an assayregarding one or more genetic loci. The sample report may also indicatea gender of an individual that provided a sample and/or indicate thatthe sample include multiple sources. As used herein, a “sample report”may include digital data (e.g., a data file) of a genetic locus orpredetermined set of genetic locus and/or a printed report of thegenetic locus or the set of genetic loci. Thus, generating or providingmay include creating a data file and/or printing the sample report, ordisplaying the sample report.

The sample report may indicate that a variant call was determined, butwas not validated. When a variant call is determined invalid, the samplereport may indicate additional information regarding the basis for thedetermination to not validate the variant call. For example, theadditional information in the report may include a description of theraw fragments and an extent (e.g., a count) to which the raw fragmentssupport or contradicted the variant call. Additionally or alternatively,the additional information in the report may include the quality scoreobtained in accordance with implementations described herein.

Variant Call Application

Implementations disclosed herein include analyzing sequencing data toidentify potential variant calls. Variant calling may be performed uponstored data for a previously performed sequencing operation.Additionally or alternatively, it may be performed in real time while asequencing operation is being performed. Each of the sample reads isassigned to corresponding genetic loci. The sample reads may be assignedto corresponding genetic loci based on the sequence of the nucleotidesof the sample read or, in other words, the order of nucleotides withinthe sample read (e.g., A, C, G, T). Based on this analysis, the sampleread may be designated as including a possible variant/allele of aparticular genetic locus. The sample read may be collected (oraggregated or binned) with other sample reads that have been designatedas including possible variants/alleles of the genetic locus. Theassigning operation may also be referred to as a calling operation inwhich the sample read is identified as being possibly associated with aparticular genetic position/locus. The sample reads may be analyzed tolocate one or more identifying sequences (e.g., primer sequences) ofnucleotides that differentiate the sample read from other sample reads.More specifically, the identifying sequence(s) may identify the sampleread from other sample reads as being associated with a particulargenetic locus.

The assigning operation may include analyzing the series of nnucleotides of the identifying sequence to determine if the series of nnucleotides of the identifying sequence effectively matches with one ormore of the select sequences. In particular implementations, theassigning operation may include analyzing the first n nucleotides of thesample sequence to determine if the first n nucleotides of the samplesequence effectively matches with one or more of the select sequences.The number n may have a variety of values, which may be programmed intothe protocol or entered by a user. For example, the number n may bedefined as the number of nucleotides of the shortest select sequencewithin the database. The number n may be a predetermined number. Thepredetermined number may be, for example, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.However, fewer or more nucleotides may be used in other implementations.The number n may also be selected by an individual, such as a user ofthe system. The number n may be based on one or more conditions. Forinstance, the number n may be defined as the number of nucleotides ofthe shortest primer sequence within the database or a designated number,whichever is the smaller number. In some implementations, a minimumvalue for n may be used, such as 15, such that any primer sequence thatis less than 15 nucleotides may be designated as an exception.

In some cases, the series of n nucleotides of an identifying sequencemay not precisely match the nucleotides of the select sequence.Nonetheless, the identifying sequence may effectively match the selectsequence if the identifying sequence is nearly identical to the selectsequence. For example, the sample read may be called for a genetic locusif the series of n nucleotides (e.g., the first n nucleotides) of theidentifying sequence match a select sequence with no more than adesignated number of mismatches (e.g., 3) and/or a designated number ofshifts (e.g., 2). Rules may be established such that each mismatch orshift may count as a difference between the sample read and the primersequence. If the number of differences is less than a designated number,then the sample read may be called for the corresponding genetic locus(i.e., assigned to the corresponding genetic locus). In someimplementations, a matching score may be determined that is based on thenumber of differences between the identifying sequence of the sampleread and the select sequence associated with a genetic locus. If thematching score passes a designated matching threshold, then the geneticlocus that corresponds to the select sequence may be designated as apotential locus for the sample read. In some implementations, subsequentanalysis may be performed to determine whether the sample read is calledfor the genetic locus.

If the sample read effectively matches one of the select sequences inthe database (i.e., exactly matches or nearly matches as describedabove), then the sample read is assigned or designated to the geneticlocus that correlates to the select sequence. This may be referred to aslocus calling or provisional-locus calling, wherein the sample read iscalled for the genetic locus that correlates to the select sequence.However, as discussed above, a sample read may be called for more thanone genetic locus. In such implementations, further analysis may beperformed to call or assign the sample read for only one of thepotential genetic loci. In some implementations, the sample read that iscompared to the database of reference sequences is the first read frompaired-end sequencing. When performing paired-end sequencing, a secondread (representing a raw fragment) is obtained that correlates to thesample read. After assigning, the subsequent analysis that is performedwith the assigned reads may be based on the type of genetic locus thathas been called for the assigned read.

Next, the sample reads are analyzed to identify potential variant calls.Among other things, the results of the analysis identify the potentialvariant call, a sample variant frequency, a reference sequence and aposition within the genomic sequence of interest at which the variantoccurred. For example, if a genetic locus is known for including SNPs,then the assigned reads that have been called for the genetic locus mayundergo analysis to identify the SNPs of the assigned reads. If thegenetic locus is known for including polymorphic repetitive DNAelements, then the assigned reads may be analyzed to identify orcharacterize the polymorphic repetitive DNA elements within the samplereads. In some implementations, if an assigned read effectively matcheswith an STR locus and an SNP locus, a warning or flag may be assigned tothe sample read. The sample read may be designated as both an STR locusand an SNP locus. The analyzing may include aligning the assigned readsin accordance with an alignment protocol to determine sequences and/orlengths of the assigned reads. The alignment protocol may include themethod described in International Patent Application No.PCT/US2013/030867 (Publication No. WO 2014/142831), filed on Mar. 15,2013, which is herein incorporated by reference in its entirety.

Then, the one or more processors analyze raw fragments to determinewhether supporting variants exist at corresponding positions within theraw fragments. Various types of raw fragments may be identified. Forexample, the variant caller may identify a type of raw fragment thatexhibits a variant that validates the original variant call. Forexample, the type of raw fragment may represent a duplex stitchedfragment, a simplex stitched fragment, a duplex un-stitched fragment ora simplex un-stitched fragment. Optionally other raw fragments may beidentified instead of or in addition to the foregoing examples. Inconnection with identifying each type of raw fragment, the variantcaller also identifies the position, within the raw fragment, at whichthe supporting variant occurred, as well as a count of the number of rawfragments that exhibited the supporting variant. For example, thevariant caller may output an indication that 10 reads of raw fragmentswere identified to represent duplex stitched fragments having asupporting variant at a particular position X. The variant caller mayalso output indication that five reads of raw fragments were identifiedto represent simplex un-stitched fragments having a supporting variantat a particular position Y. The variant caller may also output a numberof raw fragments that corresponded to reference sequences and thus didnot include a supporting variant that would otherwise provide evidencevalidating the potential variant call at the genomic sequence ofinterest.

Next, a count is maintained of the raw fragments that include supportingvariants, as well as the position at which the supporting variantoccurred. Additionally or alternatively, a count may be maintained ofthe raw fragments that did not include supporting variants at theposition of interest (relative to the position of the potential variantcall in the sample read or sample fragment). Additionally oralternatively, a count may be maintained of raw fragments thatcorrespond to a reference sequence and do not authenticate or confirmthe potential variant call. The information determined is output to thevariant call validation application, including a count and type of theraw fragments that support the potential variant call, positions of thesupporting variance in the raw fragments, a count of the raw fragmentsthat do not support the potential variant call and the like.

When a potential variant call is identified, the process outputs anindicating of the potential variant call, the variant sequence, thevariant position and a reference sequence associated therewith. Thevariant call is designated to represent a “potential” variant as errorsmay cause the call process to identify a false variant. In accordancewith implementations herein, the potential variant call is analyzed toreduce and eliminate false variants or false positives. Additionally oralternatively, the process analyzes one or more raw fragments associatedwith a sample read and outputs a corresponding variant call associatedwith the raw fragments.

Variant Classifier

FIG. 1A shows one implementation of variant calling by a trained variantclassifier disclosed herein. The trained variant classifier includes aconvolutional neural network (CNN). The input to the variant classifieris an array of input features (described with reference to FIG. 2 ). Thearray is encoded from reads (or sequence reads). Bases (or nucleotides)in reads are identified or base called through primary analysis ofsequencing data produced by genome analyzers using sequencing protocolslike sequencing-by-synthesis (SBS). Candidate variants at candidatevariant sites spanning in the reads are identified by an alignmentprocess, one implementation of which is discussed below.

Recent hardware and software improvements have resulted in a significantincrease in the data output capacity of genome analyzers such asIllumina sequencing systems (e.g., HiSegX™, HiSeg3000™, HiSeg4000™,NovaSeq 6000™, MiSegDx™, Firefly™). Greater than 33 gigabyte (GB) ofsequence output, comprising approximately 300 million 2×100 base pair(bp) reads, can now be routinely generated within 10 days. In oneimplementation, the technology disclosed uses Illumina's ConsensusAssessment of Sequence And Variation (CASAVA) software, which seamlesslyprocesses this large volume of sequencing data, supporting sequencing oflarge or small genomes, targeted deoxyribonucleic acid (DNA)resequencing, and ribonucleic acid (RNA) sequencing.

CASAVA can analyze sequencing data (e.g., image data, detection data)generated by the genome analyzers in two steps. In the first step(primary analysis), a Sequencing Control Software Real Time Analysis(SCS/RTA), which runs on an instrument computer, performs real-time dataanalysis and base calling. Base calling produces reads. In the secondstep, CASAVA performs complete secondary analysis of the reads byaligning the reads against a reference read (or reference genome) todetermine sequence differences (e.g., candidate variants likesingle-base polymorphisms (SNPs), insertions/deletions (indels)), alarger overall sequence, or the like. Algorithms for the alignment ofreads and detection of candidate variants are described in Illumina'spatent application No. WO05068089 and Illumina's technical note titled“Complete Secondary Analysis Workflow for the Genome Analyzer”(available athttps://www.illumina.com/documents/products/technotes/technote casavasecondary analysis.pdf), which are incorporated by reference as if fullyset forth herein.

In other implementations, the primary and secondary analysis areperformed by other Illumina Applications such as Whole Genome Sequencingand DRAGEN, additional details of which can be found athttps://www.illumina.com/products/by-type/informatics-products/basespace-sequence-hub/apps/whole-genome-sequencing.html?langseHus/andhttps://support.illumina.com/content/dam/illumina-marketing/documents/products/technotes/illumina-proactive-technical-note-1000000052503.pdf,which are incorporated by reference as if fully set forth herein

Array of Input Features

FIG. 2 is one implementation of the array of input features that is fedto the convolutional neural network of the variant classifier of FIG.1A. The array encodes a group of reads that are aligned to a referenceread. Each read in the group includes a target base position(highlighted in grey). The target base position corresponds to acandidate variant at a candidate variant site (e.g., SNP, indel). Thetarget base position is flanked by or padded to bases on each side(e.g., left flanking bases, right flanking bases). In someimplementations, the number of left flanking bases is the same as thenumber of right flanking bases. In other implementations, the number ofleft flanking bases is different than the number of right flankingbases. The number of flanking bases on each side can be 30, 70, 90, 110,and so on.

The group of reads is row-wise arranged in the array along the x-axis(i.e., along a first spatial dimension, e.g., height dimension), inaccordance with one implementation. That is, each row in the arrayrepresents a read that is aligned to the reference read and includes thetarget base position. Base positions in the reads are column-wisearranged in the array along the y-axis (i.e., along a second spatialdimension, e.g., width dimension), in accordance with oneimplementation. That is, each column in the array represents bases inthe reads at a particular ordinal position.

Each unit in the array is an input feature (depicted by a front-facingbox in FIG. 2 ). Each input feature in the array corresponds to a basein the reads. Each input feature in the array has a plurality ofdimensions. The plurality of dimensions is arranged in the array alongthe z-axis (e.g., along a depth, channel, feature, or fibre dimension),in accordance with one implementation.

In one implementation, the plurality of dimensions includes (i) a firstdimension set identifying the base, (ii) a second dimension setidentifying a reference base aligned to the base, (iii) a thirddimension set identifying a base call accuracy score of the base, (iv) afourth dimension set identifying strandedness (i.e., DNA strandedness)of the base, (v) a fifth dimension set identifying an insertion count(INS) of changes adjoining a position of the base, (vi) a sixthdimension set identifying a deletion flag (DEL) at the position of thebase.

In other implementations, the array can be considered a volume. In yetother implementations, the array can be considered a tensor. In someimplementations, the array represents a read pileup around a candidatevariant. In some implementations, the dimensions of an input feature canbe considered input channels.

In one example, each input feature has twelve dimensions. Then, thefirst dimension set includes four dimensions that use one-hot encodingto identify the base of the input features. The base can be Adenine (A),Cytosine (C), Guanine (G), or Thymine (T). The second dimension set alsoincludes four dimensions that use one-hot encoding to identify thereference base aligned to the base. The reference base can also be A, C,G, or T.

In one-hot encoding, each base in a sequence is encoded with a binaryvector of four bits, with one of the bits being hot (i.e., 1) whileother being 0. For instance, A=(1, 0, 0, 0), C=(0, 1, 0, 0), G=(0, 0, 1,0), and T=(0, 0, 0, 1). In some implementations, an unknown base isencoded as N=(0, 0, 0, 0).

Accordingly, each input feature “locally” encodes alignment between thebase in a read and the corresponding reference base in the referenceread. As a result, when kernels of convolution filters of theconvolutional neural network of the variant classifier of FIG. 1A areapplied over a window of input features in the array, they take intoaccount so-called “one-on-one contextual dependencies” between bases inthe reference read and bases in the reads, as well as so-called“adjacent contextual dependencies” between bases in the reads.

The third, fourth, fifth, and sixth dimension sets each include onedimension to respectively identify the base call accuracy score of thebase as a continuous number, the strandedness of the base using one-hotencoding (e.g., 0 for forward strand and 1 for reverse strand), theinsertion count (INS) of changes adjoining a position of the base asnumbers (e.g., 4 for 4 inserted bases), and the deletion flag (DEL) atthe position of the base as numbers (e.g., 1111 for 4 deleted basepositions). In FIG. 2 , the six dimension sets of an input feature aregraphically distinguished using different shades of grey.

In some implementations, the mapping quality of each read is alsoencoded in the array. The mapping quality (MAPA) is a number (e.g., 40)that can be encoded in an additional dimension or channel of each unitor each input feature in the array.

Regarding the base call accuracy score, in one implementation, it can beidentified as a Phred quality score (e.g., Q10, Q20, Q30, Q40, Q50)defined as property that is logarithmically related to the base callingerror probabilities (P)². Additional information about the base callaccuracy score can be found in Illumina's technical notes titled“Quality Scores for Next-Generation Sequencing” and “UnderstandingIllumina Quality Scores” (available athttps://www.illumina.com/documents/products/technotes/technoteQ-Scores.pdf,https://www.illumina.com/documents/products/technotes/technoteunderstanding quality scores .pdf), which are incorporated by referenceas if fully set forth herein.

Regarding the insertion count (INS) of changes adjoining a position ofthe base, in one implementation, it can identify a number of basesinserted before or after the base. Regarding the deletion flag (DEL) atthe position of the base, in one implementation, it can identify anundetermined, unread, unidentified, empty, or deleted base at theposition of the base.

In one implementation, the dimensionality of the array is 100×221×12,where: (a) 100 represents the number of reads in the group that arealigned to the reference read and span the candidate variant sites atthe target base position; (b) 221 represents the number of basepositions in each of the reads, with the target base position at the111^(th) ordinal position flanked by 110 base positions on each side;and (c) 12 represents the local dimensionality of each input feature inthe array, i.e., the number of dimensions of each of the input features.

In other implementations, the input features can have different numbersof dimensions, which can be further segmented into dimension sets ofvarying sizes using a different encoding scheme.

In yet other implementations, one-hot encoding may be replaced by otherencoding schemes such as a dense or real-valued encoding scheme based onan embedding space or embedding matrix produced by a trained neuralnetwork. In yet further implementations, the encoding schemes can bebased on quantitative or numerical data type, qualitative data type,discreet data type, continuous data type (with lower and upper bounds),integer data type (with lower and upper bounds), nominal data type,ordinal or ranked data type, categorical data type, interval data type,and/or ratio data type. For example, the encoding can be based on, orany combination thereof, real values between 0 and 1, continuous valuessuch as red, green, blue (RGB) values between 0 and 256, hexadecimalvalues, size of a particular dimension (e.g., height and width), a setof different values and data types, and others.

Variant Classifier CNN Architecture

As discussed above, the array of input features that is fed to theconvolutional neural network of the variant classifier of FIG. 1A. FIG.3A illustrates one implementation of architecture 300A of theconvolutional neural network of the variant classifier of FIG. 1A.Specifically, the convolutional neural network architecture illustratedin FIG. 3A has eight convolution layers. The variant classifierconvolutional neural network can include an input layer that is followedby a plurality of convolution layers. Some of the convolution layers canbe followed by a max pooling (or sampling) layer, with an intermediatebatch normalization layer between the convolution layer and the maxpooling layer. In the illustrated implementation, the convolutionalneural network has eight convolution layers, three max pooling layers,and eight batch normalization layers.

Regarding batch normalization, batch normalization is a method foraccelerating deep network training by making data standardization anintegral part of the network architecture. Batch normalization canadaptively normalize data even as the mean and variance change over timeduring training. It works by internally maintaining an exponentialmoving average of the batch-wise mean and variance of the data seenduring training. The main effect of batch normalization is that it helpswith gradient propagation—much like residual connections—and thus allowsfor deep networks. Some very deep networks can only be trained if theyinclude multiple Batch Normalization layers.

Batch normalization can be seen as yet another layer that can beinserted into the model architecture, just like the fully connected orconvolutional layer. The BatchNormalization layer is typically usedafter a convolutional or densely connected layer. It can also be usedbefore a convolutional or densely connected layer. Both implementationscan be used by the technology disclosed. The BatchNormalization layertakes an axis argument, which specifies the feature axis that should benormalized. This argument defaults to −1, the last axis in the inputtensor. This is the appropriate value when using Dense layers, ConvlDlayers, RNN layers, and Conv2D layers with data_format set to“channels_last”. But in the niche use case of Conv2D layers withdata_format set to “channels_first”, the features axis is axis 1; theaxis argument in BatchNormalization can be set to 1.

Batch normalization provides a definition for feed-forwarding the inputand computing the gradients with respect to the parameters and its owninput via a backward pass. In practice, batch normalization layers areinserted after a convolutional or fully connected layer, but before theoutputs are fed into an activation function. For convolutional layers,the different elements of the same feature map—i.e. the activations—atdifferent locations are normalized in the same way in order to obey theconvolutional property. Thus, all activations in a mini-batch arenormalized over all locations, rather than per activation.

The internal covariate shift is the major reason why deep architectureshave been notoriously slow to train. This stems from the fact that deepnetworks do not only have to learn a new representation at each layer,but also have to account for the change in their distribution.

The covariate shift in general is a known problem in the deep learningdomain and frequently occurs in real-world problems. A common covariateshift problem is the difference in the distribution of the training andtest set which can lead to suboptimal generalization performance. Thisproblem is usually handled with a standardization or whiteningpreprocessing step. However, especially the whitening operation iscomputationally expensive and thus impractical in an online setting,especially if the covariate shift occurs throughout different layers.

The internal covariate shift is the phenomenon where the distribution ofnetwork activations change across layers due to the change in networkparameters during training. Ideally, each layer should be transformedinto a space where they have the same distribution but the functionalrelationship stays the same. In order to avoid costly calculations ofcovariance matrices to decorrelate and whiten the data at every layerand step, we normalize the distribution of each input feature in eachlayer across each mini-batch to have zero mean and a standard deviationof one.

During the forward pass, the mini-batch mean and variance arecalculated. With these mini-batch statistics, the data is normalized bysubtracting the mean and dividing by the standard deviation. Finally,the data is scaled and shifted with the learned scale and shiftparameters. Since normalization is a differentiable transform, theerrors are propagated into these learned parameters and are thus able torestore the representational power of the network by learning theidentity transform. Conversely, by learning scale and shift parametersthat are identical to the corresponding batch statistics, the batchnormalization transform would have no effect on the network, if that wasthe optimal operation to perform. At test time, the batch mean andvariance are replaced by the respective population statistics since theinput does not depend on other samples from a mini-batch. Another methodis to keep running averages of the batch statistics during training andto use these to compute the network output at test time.

The convolution layers can be parametrized by a number of convolutionfilters (e.g., thirty-two filters) and a convolution window size. Theconvolution filters can be further parameterized by two spatialdimensions, namely, height and width (e.g., 5×5 or 5×1) and by a thirddepth, feature, or fibre dimension (e.g., 12, 10, 32). Inimplementations, the depth dimensionality of the convolution filters ofthe first convolution layer of the convolutional neural network matchesthe number of dimensions of the input features of the array.

The convolutional neural network can also include one or morefully-connected layers. In the illustrated embodiment, the convolutionalneural network includes two fully-connected layers. In implementations,the convolutional neural network processes the group of reads throughthe convolution layers and concatenates output of the convolution layerswith corresponding empirical variant score (EVS) features provided by asupplemental input layer. The supplemental input layer of theconvolutional neural network can be different from the input layer thatprovides the array as input to the first convolution layer of theconvolutional neural network. In one implementation, the output of thelast convolution layer of the convolutional neural network is flattenedby a flattening layer of the convolutional neural network and thencombined with the EVS features.

Regarding the EVS features, a set of EVS features can be associated withthe candidate variant site in the array (e.g., twenty three EVS featuresfor SNPs and twenty two EVS features for indels). Some examples of theEVS features include germline features, RNA-seq features, and Somaticfeatures, Germline SNV features, Germline Indel features, RNA-seq SNVfeatures, RNA-seq Indel features, Somatic SNV features, and SomaticIndel features. Additional examples of the EVS features are providedlater in this application under the Section titled “EVS Feature”.

Each EVS feature is a number that represents a specific attribute of acandidate variant site. Thus, a set of EVS features of a candidatevariant site is identified by a vector of numbers or numericaldescriptors, according to one implementation. The EVS feature numbersare fed directly to the convolutional neural network. For instance,GenotypeCategory is 0 for heterozygous sites, 1 for homozygous sites,and 2 for alt-heterozygous sites. Others, like SampleRMSMappingQualityare floating point numbers. RMS stands for Root-Mean Square EVS featureand is determined by summing the squared mapping qualities for each readcovering the site, dividing it by the number of reads, and then takingthe square root of the results of the division. We observe higheraccuracy with the ConservativeGenotypeQuality EVS feature.

After the output of the last convolution layer is concatenated with theEVS features, the convolutional neural network then feeds the result ofthe concatenation to the fully-connected layers. A classification layer(e.g., softmax layer) following the full-connected layers can produceclassification scores for likelihood that each candidate variant at thetarget base position is a true variant or a false variant. In otherimplementations, the classification layer can produce classificationscores for likelihood that each candidate variant at the target baseposition is a homozygous variant, a heterozygous variant, a non-variant,or a complex-variant.

FIG. 3B illustrates another implementation of the architecture 300B ofthe convolutional neural network of the variant classifier of FIG. 1A.FIG. 3B also shows the dimensionality of the input/output at variousprocessing phases of the convolutional neural network. Specifically, theconvolutional neural network architecture illustrated in FIG. 3B hasseven convolution layers. In this example architecture, thedimensionality of the output produced by a first 5×5 convolution layerwith thirty-two filters and a first successive max pooling layer can be108×48×32; the dimensionality of the output produced by a second 5×5convolution layer with thirty-two filters and a second successive maxpooling layer can be 52×22×32; and the dimensionality of the outputproduced by a third 5×5 convolution layer with thirty-two filters and athird successive max pooling layer can be 24×9×32. Moving ahead, thedimensionality of the output produced by a fourth 5×5 convolution layerwith thirty-two filters and no successive max pooling layer can be20×5×32; the dimensionality of the output produced by a fifth 5×5convolution layer with thirty-two filters and no successive max poolinglayer can be 16×1×32; the dimensionality of the output produced by asixth 5×1 convolution layer with thirty-two filters and no successivemax pooling layer can be 11×1×32; and the dimensionality of the outputproduced by a seventh 5×1 convolution layer with thirty-two filters andno successive max pooling layer can be 7×1×32. Moving ahead, the 7×1×32output can be flattened into a 224 dimensional vector and furtherconcatenated with a 23 or 22 dimensional EVS feature vector to produce a247 or 246 dimensional concatenated vector. The concatenated vector canbe fed to a fully-connected layers with 256 units and then to aclassification layer to produce the classification scores.

FIG. 3C illustrates yet another implementation of the architecture 300Cof the convolutional neural network of the variant classifier of FIG.1A. Specifically, the convolutional neural network architectureillustrated in FIG. 3C has five convolution layers. In this examplearchitecture, the variant classifier convolutional neural network caninclude an input layer that is followed by five 3×3 convolution layerswith thirty-two convolution filters each. Each convolution layer can befollowed by a batch normalization layer and a 2×2 max pooling layer. Theconvolutional neural network can further include a flattening layer, asupplemental input layer, a concatenation layer, two fully-connected(FC) layers, and a classification layer. FIG. 3C also shows thedimensionality of the input/output at various processing phases of theconvolutional neural network.

FIG. 3D illustrates yet another implementation of the architecture 300Dof the convolutional neural network of the variant classifier of FIG.1A. Specifically, the convolutional neural network architectureillustrated in FIG. 3D uses depthwise separable convolutions. Incontrast to a standard convolution, a depthwise separable convolutionperforms a separate convolution of each channel of the input data andthen performs a pointwise convolution to mix the channels. Foradditional information about the depthwise separable convolutions,reference can be made to A. G. Howard, M. Zhu, B. Chen, D. Kalenichenko,W. Wang, T. Weyand, M. Andreetto, and H. Adam, “Mobilenets: EfficientConvolutional Neural Networks for Mobile Vision Applications,” inarXiv:1704.04861, 2017, which is incorporated by reference as if fullyset forth herein.

Variant Classifier FC Network Architecture

FIG. 4A depicts a fully-connected (FC) network 400A in which computationunits have full connections to all the computation units of the previouslayer. Suppose that a layer has m computation units and the previouslayer gives n outputs, then we get a total number of m*n weights.

FIG. 4B illustrates one implementation of architecture 400B of thefully-connected neural network of the variant classifier, without anyconvolution layers. Architecture 400B uses fully-connected layers (alsocalled “dense layers”). In FIG. 4B, there are seven dense layers,interspersed with batch normalization and dropout layers.

In one implementation, the fully-connected neural network of the variantclassifier has four fully-connected layers, with 64 units per layer, 10%dropout rate, and a batch normalization layer after each fully-connectedlayer.

The input to the fully-connected neural network are empirical variantscore (EVS) features of a candidate variant site. Each EVS feature is anumber that represents a specific attribute of a candidate variant site.Thus, a set of EVS features of a candidate variant site is identified bya vector of numbers or numerical descriptors, according to oneimplementation. The EVS feature numbers are fed directly to theconvolutional neural network. For instance, GenotypeCategory is 0 forheterozygous sites, 1 for homozygous sites, and 2 for alt-heterozygoussites. Others, like SampleRMSMappingQuality are floating point numbers.RMS stands for Root-Mean Square EVS feature and is determined by summingthe squared mapping qualities for each read covering the site, dividingit by the number of reads, and then taking the square root of theresults of the division. We observe higher accuracy with theConservativeGenotypeQuality EVS feature.

The input to the fully-connected neural network can be any combinationof the EVS feature listed below. That is, an EVS feature vector for aparticular candidate variant site being evaluated by the variant callercan be encoded or constructed to include number values for any of theEVS features listed below.

EVS Features

The following lists examples of the EVS features under four categories:

-   -   (1) Germline SNV features: GenotypeCategory,        SampleRMSMappingQuality, SiteHomopolymerLength,        SampleStrandBias, SampleRMSMappingQualityRankSum,        SampleReadPosRankSum, RelativeTotalLocusDepth,        SampleUsedDepthFraction, ConservativeGenotypeQuality,        NormalizedAltHaplotypeCountRatio.    -   (2) Germline Indel features: GenotypeCategory,        SampleIndelRepeatCount, SampleIndelRepeatUnitSize,        SampleIndelAlleleBiasLower, SampleIndelAlleleBias,        SampleProxyRMSMappingQuality, RelativeTotalLocusDepth,        SamplePrimaryAltAlleleDepthFraction,        ConservativeGenotypeQuality, InterruptedHomopolymerLength,        ContextCompressability, IndelCategory,        NormalizedAltHaplotypeCountRatio.    -   (3) Somatic SNV features:        SomaticSNVQualityAndHomRefGermlineGenotype, Normal        SampleRelativeTotalLocusDepth, TumorSampleAltAlleleFraction,        RMSMappingQuality, ZeroMappingQualityFraction,        TumorSampleStrandBias, TumorSampleReadPosRankSum,        AlleleCountLogOddsRatio, Normal SampleFilteredDepthFraction,        TumorSampleFilteredDepthFraction.    -   (4) Somatic Indel features:        SomaticIndelQualityAndHomRefGermlineGenotype,        TumorSampleReadPosRankSum,        TumorSampleLogSymmetricStrandOddsRatio, RepeatUnitLength,        IndelRepeatCount, RefRepeatCount, InterruptedHomopolymerLength,        TumorSampleIndelNoiseLogOdds, TumorNormalIndelAlleleLogOdds,        AlleleCountLogOddsRatio.

The following are definitions of the EVS features listed above:

Germline Feature Descriptions:

GenotypeCategory A category variable reflecting the most likely genotypeas heterozygous (0), homozygous (1) or alt-heterozygous (2).SampleRMSMappingQuality RMS mapping quality of all reads spanning thevariant in one sample. This feature matches SAMPLE/MQ in the VCF spec.SiteHomopolymerLength Length of the longest homopolymer containing thecurrent position if this position can be treated as any base.InterruptedHomopolymerLength One less than the length of the longestinterrupted homopolymer in the reference sequence containing the currentposition. An interrupted homopolymer is a string that has edit distance1 to a homopolymer. SampleStrandBias Log ratio of the sample's genotypelikelihood computed assuming the alternate allele occurs on only onestrand vs both strands (thus positive values indicate bias).SampleRMSMapping Z-score of Mann-Whitney U test for QualityRankSumreference vs alternate allele mapping quality values in one sample.SampleReadPosRankSum Z-score of Mann-Whitney U test for reference vsalternate allele read positions in one sample. RelativeTotalLocusDepthLocus depth relative to expectation: this is the ratio of total readdepth at the variant locus in all samples over the total expected depthin all samples. Depth at the variant locus includes reads at any mappingquality. Expected depth is taken from the preliminary depth estimationstep. This value is set to 1 in exome and targeted analyses, because itis problematic to define expected depth in this case.SampleUsedDepthFraction The ratio of reads used to genotype the locusover the total number of reads at the variant locus in one sample. Readsare not used if the mapping quality is less than the minimum threshold,if the local read alignment fails the mismatch density filter or if thebasecall is ambiguous. ConservativeGenotypeQuality The model-basedConservativeGenotypeQuality (GQX) value for one sample, reflecting theconservative confidence of the called genotype. NormalizedAltHaplotypeFor variants in an active region, the CountRatio proportion of readssupporting the top 2 haplotypes, or 0 if haplotyping failed due to thisproportion being below threshold. For heterozygous variants with onlyone non-reference allele, the proportion is doubled so that its value isexpected to be close to 1.0 regardless of genotype. The feature is setto −1 for variants not in an active region. SampleIndelRepeatCount Thenumber of times the primary indel allele's repeat unit occurs in ahaplotype containing the indel allele. The primary indel allele's repeatunit is the smallest possible sequence such that the inserted/deletedsequence can be formed by concatenating multiple copies of it. Theprimary indel allele is the best supported allele among all overlappingindel alleles at the locus of interest in one sample.SampleIndelRepeatUnitSize Length of the primary indel allele's repeatunit, as defined for feature SampleIndelRepeatCount.SampleIndelAlleleBiasLower The negative log probability of seeing N orfewer observations of one allele in a heterozygous variant out of thetotal observations from both alleles in one sample. N is typically theobservation count of the reference allele. If the heterozygous variantdoes not include the reference allele, the first indel allele is usedinstead. SampleIndelAlleleBias Similar to SampleIndelAlleleBiasLower,except the count used is twice the count of the least frequentlyobserved allele. SampleProxyRMS RMS mapping quality of all readsMappingQuality spanning the position immediately preceding the indel inone sample. This feature approximates the SAMPLE/MQ value defined in theVCF spec. SamplePrimaryAltAllele The ratio of the confident observationDepthFraction count of the best-supported non- reference allele at thevariant locus, over all confident allele observation counts in onesample. ContextCompressability The length of the upstream or downstreamreference context (whichever is greater) that can be represented using 5Ziv-Lempel keywords. The Ziv-Lempel keywords are obtained using thescheme of Ziv and Lempel 1977, by traversing the sequence andsuccessively selecting the shortest subsequence that has not yet beenencountered. IndelCategory A binary variable set to 1 if the indelallele is a primitive deletion or 0 otherwise.SamplePrimaryAltAlleleDepth The confident observation count of thebest-supported non-reference allele at the variant locus.VariantAlleleQuality The model-based variant quality value reflectingconfidence that the called variant is present in at least one sample,regardless of genotype. This feature matches QUAL in the VCF spec.SampleMeanDistance For all non-reference base call FromReadEdgeobservations in one sample at a candidate SNV site, report the meandistance to the closest edge of each alternate base call's read.Distance is measured in read-coordinates, zero- indexed, and is allowedto have a maximum value of 20. SampleRefAlleleDepth The confidentobservation count of the reference allele at the variant locus.SampleIndelMeanDistance For all indel allele observations in oneFromReadEdge sample at a candidate indel locus, report the mean distanceto the closest edge of each indel allele's read. Distance is measured inread-coordinates, zero- indexed, and is allowed to have a maximum valueof 20. The left or right side of the indel may be used to provide theshortest distance, but the indel will only be considered in itsleft-aligned position. SampleRefRepeatCount The number of times theprimary indel allele's repeat unit occurs in the reference sequence.

Somatic Feature Descriptions:

Note that for somatic features “all samples” refers to the tumor andmatched normal samples together.

SomaticSNVQualityAndHomRefGermlineGenotype Posterior probability of asomatic SNV conditioned on a homozygous reference germline genotype.When INFO/NT is “ref”, this feature matches INFO/QSS_NT in the VCFoutput. NormalSampleRelativeTotalLocusDepth This feature matches thegermline RelativeTotalLocusDepth feature, except that it reflects thedepth of only the matched normal sample. TumorSampleAltAlleleFractionFraction of the tumor sample's observations which are not the referenceallele. This is restricted to a maximum of 0.5 to prevent the model fromovertraining against high somatic allele frequencies (these might becommon e.g. for loss of heterozygosity regions from liquid tumors).RMSMappingQuality Root mean square read mapping quality of all readsspanning the variant in all samples. This feature matches INFO/MQ in theVCF spec. ZeroMappingQualityFraction Fraction of read mapping qualitiesequal to zero, for all reads spanning the variant in all samples.InterruptedHomopolymerLength One less than the length of the longestinterrupted homopolymer in the reference sequence containing the currentposition. An interrupted homopolymer is a string that has edit distance1 to a homopolymer. TumorSampleStrandBias Log ratio of the tumor-samplesomatic allele likelihood computed assuming the somatic allele occurs ononly one strand vs both strands (thus higher values indicate greaterbias). TumorSampleReadPosRankSum Z-score of Mann-Whitney U test forreference vs non-reference allele read positions in the tumor sample'sobservations. AlleleCountLogOddsRatio The log odds ratio of allelecounts${\log\frac{r_{t}a_{n}}{r_{n}{at}}},{{given}{reference}\left( {r_{t},r_{n}} \right){and}}$non-reference (a_(t), a_(n)) allele counts for the tumor and normalsample pair. NormalSampleFilteredDepthFraction The fraction of readsthat were filtered out of the normal sample before calling the variantlocus. TumorSampleFilteredDepthFraction The fraction of reads that werefiltered out of the tumor sample before calling the variant locus.SomaticIndelQualityAndHomRefGermlineGenotype Posterior probability of asomatic indel conditioned on a homozygous reference germline genotype.When INFO/NT is “ref”, this feature matches INFO/QSI_NT in the VCFoutput. TumorSampleLogSymmetricStrandOddsRatio Log of the symmetricstrand odds ratio${{of}{allele}{counts}\log\left( {\frac{r_{fwd}a_{rev}}{r_{rev}a_{fwd}} + \frac{r_{rev}a_{fwd}}{r_{fwd}a_{rev}}} \right)},$given reference (r_(fwd), r_(rev)) and non- reference (a_(fwd), a_(rev))confident counts of the tumor sample's observations. RepeatUnitLengthThe length of the somatic indel allele's repeat unit. The repeat unit isthe smallest possible sequence such that the inserted/deleted sequencecan be formed by concatenating multiple copies of it. IndelRepeatCountThe number of times the somatic indel allele's repeat unit occurs in ahaplotype containing the indel allele. RefRepeatCount The number oftimes the somatic indel allele's repeat unit occurs in the referencesequence. TumorSampleIndelNoiseLogOdds Log ratio of the frequency of thecandidate indel vs all other indels at the same locus in the tumorsample. The frequencies are computed from reads which confidentlysupport a single allele at the locus. TumorNormalIndelAlleleLogOdds Logratio of the frequency of the candidate indel in the tumor vs normalsamples. The frequencies are computed from reads which confidentlysupport a single allele at the locus. SiteFilteredBasecallFrac Themaximum value over all samples of SampleSiteFilteredBasecallFrac, whichis the fraction of base calls at a site which have been removed by themismatch density filter in a given sample.IndelWindowFilteredBasecallFrac The maximum value over all samples ofSampleIndelWindowFilteredBasecallFrac, which is the fraction of basecalls in a window extending 50 bases to each side of the candidateindel's call position which have been removed by the mismatch densityfilter in a given sample. SpanningDeletionFraction The maximum valueover all samples of SampleSpanningDeletionFraction, which is thefraction of reads crossing a candidate SNV site with spanning deletionsin a given sample.

In some implementations, the input includes only EVS features. In otherimplementations, in the input, the EVS features can be supplemented byread data, as discussed above with the CNN implementations.

FIG. 1B illustrates one implementation of training the variantclassifier of FIG. 1A using labeled training data comprising candidatevariants (SNPs and indels). The variant classifier is trained on fiftythousand (50000) to one million (1000000) candidate variants (SNPs andindels) in various implementations. The candidate variants are labeledwith true variant classifications and thus serve as the ground truthduring the training. In one implementation, one million trainingexamples of candidate variant sites with 50 to 100 reads each can betrained on a single GPU card in less than 10 hours with good recall andprecision over 5-10 epochs of training. Training data can includeNA129878 samples, with validation data from chromosome 2/20 held out.The variant classifier convolutional neural network is trained usingbackpropagation-based stochastic gradient descent algorithms such asAdam and regularization techniques like Dropout.

FIG. 1C depicts one implementation of input and output modules ofconvolutional neural network processing of the variant classifier ofFIG. 1A. The input module includes feed the array of input features tothe convolutional neural network, as discussed above. The output moduleincludes translating analysis by the convolutional neural network intoclassification scores for likelihood that each candidate variant at thetarget base position is a true variant or a false variant. A finalsoftmax classification layer of the convolutional neural network canproduce normalized probabilities for the two classes that add up tounity (1). In the illustrated example, the softmax probability of thetrue positive (or true variant) is 0.85 and the softmax probability ofthe false positive (or false variant) is 0.15. Consequently, thecandidate variant at the target base position is classified as a truevariant.

For additional information about the architecture, training, inference,analysis, and translation of the variant classifier convolutional neuralnetwork, reference can be made to J. Wu, “Introduction to ConvolutionalNeural Networks,” Nanjing University, 2017; I. J. Goodfellow, D.Warde-Farley, M. Mirza, A. Courville, and Y. Bengio, “CONVOLUTIONALNETWORKS”, Deep Learning, MIT Press, 2016; and “BATCH NORMALIZATION:ACCELERATING DEEP NETWORK TRAINING BY REDUCING INTERNAL COVARIATESHIFT,” arXiv: 1502.03167, 2015, which are incorporated by reference asif fully set forth herein.

In yet other implementations, the convolutional neural network of thevariant classifier of FIG. 1A can use 1D convolutions, 2D convolutions,3D convolutions, 4D convolutions, 5D convolutions, dilated or atrousconvolutions, transpose convolutions, depthwise separable convolutions,pointwise convolutions, 1×1 convolutions, group convolutions, flattenedconvolutions, spatial and cross-channel convolutions, shuffled groupedconvolutions, spatial separable convolutions, and deconvolutions. It canuse one or more loss functions such as logistic regression/log loss,multi-class cross-entropy/softmax loss, binary cross-entropy loss,mean-squared error loss, L1 loss, L2 loss, smooth L1 loss, and Huberloss. It can use any parallelism, efficiency, and compression schemessuch TFRecords, compressed encoding (e.g., PNG), sharding, parallelcalls for map transformation, batching, prefetching, model parallelism,data parallelism, and synchronous/asynchronous SGD. It can includeupsampling layers, downsampling layers, recurrent connections, gates andgated memory units (like an LSTM or GRU), residual blocks, residualconnections, highway connections, skip connections, activation functions(e.g., non-linear transformation functions like rectifying linear unit(ReLU), leaky ReLU, exponential liner unit (ELU), sigmoid and hyperbolictangent (tanh)), batch normalization layers, regularization layers,dropout, pooling layers (e.g., max or average pooling), global averagepooling layers, and attention mechanisms.

Experimental Results

FIG. 5 shows one example of precision-recall curves that comparesingle-base polymorphism (SNP) classification performance by theconvolutional neural network of the variant classifier and by a baselineStrelka™ model called empirical variant score (EVS) model. As shown inFIG. 5 , the convolutional neural network of the variant classifier hasbetter precision-recall for SNPs than the EVS model.

FIG. 6 shows another example of precision-recall curves that compare SNPclassification performance by the convolutional neural network of thevariant classifier and by the EVS model. Here, the convolutional neuralnetwork of the variant classifier is trained on a larger training setand thus further outperforms the EVS model.

FIG. 7 depicts one example of precision-recall curves that compare indelclassification performance by the convolutional neural network of thevariant classifier and by the EVS model. As shown in FIG. 7 , theconvolutional neural network of the variant classifier has betterprecision-recall for indels than the EVS model.

FIG. 8 illustrates convergence curves of the convolutional neuralnetwork of the variant classifier during training and validation. Asshown in FIG. 8 , the convolutional neural network converges around 8-9epochs during training and validation, with each epoch taking around onehour to complete on a single GPU.

FIG. 9 illustrates convergence curves of the fully-connected neuralnetwork of the variant classifier during training and testing(inference). As shown in FIG. 9 , the fully-connected neural networkconverges after 14 epochs during training and testing.

In other implementations, the variant classifier can be trained for 50epochs, with small improvements after 20 to 30 epochs withoutoverfitting.

FIG. 10 uses precision-recall curves to compare SNP classificationperformance of (i) the fully-connected neural network of the variantclassifier trained on EVS features of the EVS model version 2.8.2, (ii)the fully-connected neural network of the variant classifier trained onEVS features of the EVS model version 2.9.2, (iii) the EVS model version2.8.2, and (iv) the EVS model version 2.9.2. As shown in FIG. 10 , thefully-connected neural networks of the variant classifier outperform theEVS models.

FIG. 11 uses precision-recall curves to compare indel classificationperformance of (i) the fully-connected neural network of the variantclassifier trained on EVS features of the EVS model version 2.8.2, (ii)the fully-connected neural network of the variant classifier trained onEVS features of the EVS model version 2.9.2, (iii) the EVS model version2.8.2, and (iv) the EVS model version 2.9.2. As shown in FIG. 11 , thefully-connected neural networks of the variant classifier outperform theEVS models.

Computer System

FIG. 12 is a simplified block diagram of a computer system that can beused to implement the variant classifier. Computer system 1200 includesat least one central processing unit (CPU) 1272 that communicates with anumber of peripheral devices via bus subsystem 1255. These peripheraldevices can include a storage subsystem 1210 including, for example,memory devices and a file storage subsystem 1236, user interface inputdevices 1238, user interface output devices 1276, and a networkinterface subsystem 1274. The input and output devices allow userinteraction with computer system 1200. Network interface subsystem 1274provides an interface to outside networks, including an interface tocorresponding interface devices in other computer systems.

In one implementation, the variant classifier is communicably linked tothe storage subsystem 1210 and the user interface input devices 1238.

User interface input devices 1238 can include a keyboard; pointingdevices such as a mouse, trackball, touchpad, or graphics tablet; ascanner; a touch screen incorporated into the display; audio inputdevices such as voice recognition systems and microphones; and othertypes of input devices. In general, use of the term “input device” isintended to include all possible types of devices and ways to inputinformation into computer system 1200.

User interface output devices 1276 can include a display subsystem, aprinter, a fax machine, or non-visual displays such as audio outputdevices. The display subsystem can include an LED display, a cathode raytube (CRT), a flat-panel device such as a liquid crystal display (LCD),a projection device, or some other mechanism for creating a visibleimage. The display subsystem can also provide a non-visual display suchas audio output devices. In general, use of the term “output device” isintended to include all possible types of devices and ways to outputinformation from computer system 1200 to the user or to another machineor computer system.

Storage subsystem 1210 stores programming and data constructs thatprovide the functionality of some or all of the modules and methodsdescribed herein. These software modules are generally executed by deeplearning processors 1278.

Deep learning processors 1278 can be graphics processing units (GPUs),field-programmable gate arrays (FPGAs), application-specific integratedcircuits (ASICs), and/or coarse-grained reconfigurable architectures(CGRAs). Deep learning processors 1278 can be hosted by a deep learningcloud platform such as Google Cloud Platform™, Xilinx™, and Cirrascale™.Examples of deep learning processors 1278 include Google's TensorProcessing Unit (TPU)™, rackmount solutions like GX4 Rackmount Series™,GX12 Rackmount Series™ NVIDIA DGX-1™, Microsoft' Stratix V FPGA™,Graphcore's Intelligent Processor Unit (IPU)™, Qualcomm's ZerothPlatform™ with Snapdragon Processors™, NVIDIA's Volta™ NVIDIA's DRIVEPX™, NVIDIA's JETSON TX1/TX2 MODULE™, Intel's Nirvana™ Movidius VPU™,Fujitsu DPI™, ARM's DynamicIQ™, IBM TrueNorth™, and others.

Memory subsystem 1222 used in the storage subsystem 1210 can include anumber of memories including a main random access memory (RAM) 1232 forstorage of instructions and data during program execution and a readonly memory (ROM) 1234 in which fixed instructions are stored. A filestorage subsystem 1236 can provide persistent storage for program anddata files, and can include a hard disk drive, a floppy disk drive alongwith associated removable media, a CD-ROM drive, an optical drive, orremovable media cartridges. The modules implementing the functionalityof certain implementations can be stored by file storage subsystem 1236in the storage subsystem 1210, or in other machines accessible by theprocessor.

Bus subsystem 1255 provides a mechanism for letting the variouscomponents and subsystems of computer system 1200 communicate with eachother as intended. Although bus subsystem 1255 is shown schematically asa single bus, alternative implementations of the bus subsystem can usemultiple busses.

Computer system 1200 itself can be of varying types including a personalcomputer, a portable computer, a workstation, a computer terminal, anetwork computer, a television, a mainframe, a server farm, awidely-distributed set of loosely networked computers, or any other dataprocessing system or user device. Due to the ever-changing nature ofcomputers and networks, the description of computer system 1200 depictedin FIG. 12 is intended only as a specific example for purposes ofillustrating the preferred embodiments of the present invention. Manyother configurations of computer system 1200 are possible having more orless components than the computer system depicted in FIG. 12 .

Particular Implementations

Convolutional Neural Network (CNN) Implementations

The technology disclosed relates to a system comprising a trainedvariant classifier. The variant classifier includes numerous processorsoperating in parallel and coupled to memory. The variant classifier alsoincludes a convolutional neural network that runs on the numerousprocessors.

The convolutional neural network is trained on at least 50000 to 1000000training examples of groups of reads that span candidate variant sitesand are labeled with true variant classifications of the groups. Each ofthe training examples used in the training includes a group of readsthat are aligned to a reference read. Each of the reads includes atarget base position that is flanked by or padded to at least 110 baseson each side. Each of the bases in the reads is accompanied by acorresponding reference base in the reference read, a base call accuracyscore of reading the base, a strandedness (i.e., DNA strandedness) ofreading the base, insertion count of changes adjoining a position of thebase, and deletion flag at the position of the base.

An input module of the convolutional neural network, which runs on atleast one of the numerous processors, feeds the group of reads forevaluation of the target base position.

An output module of the convolutional neural network, which runs on atleast one of the numerous processors, translates analysis by theconvolutional neural network into classification scores for likelihoodthat each candidate variant at the target base position is a truevariant or a false variant.

This system implementation and other systems disclosed optionallyinclude one or more of the following features. System can also includefeatures described in connection with methods disclosed. In the interestof conciseness, alternative combinations of system features are notindividually enumerated. Features applicable to systems, methods, andarticles of manufacture are not repeated for each statutory class set ofbase features. The reader will understand how features identified inthis section can readily be combined with base features in otherstatutory classes.

The convolutional neural network can have one or more convolution layersand one or more fully-connected layers. The convolutional neural networkcan process the group of reads through the convolution layers andconcatenate output of the convolution layers with correspondingempirical variant score (abbreviated EVS) features. The convolutionalneural network can further feed the result of the concatenation to thefully-connected layers.

The bases in the reads can be encoded using one-hot encoding. Thecorresponding base in the reference read can be encoded using one-hotencoding. The base call accuracy score of reading the base can beencoded as a continuous number. The strandedness of reading the base canbe encoded using one-hot encoding. The insertion count of changesadjoining the position of the base can be encoded as a number. Thedeletion flag at the position of the base can be encoded as a number.

The candidate variant can be a candidate single-base polymorphism(abbreviated SNP). The candidate variant can be a candidate insertion ordeletion (abbreviated indel).

The numerous processors can be part of a graphics processing unit(abbreviate GPU). The convolutional neural network can run on the GPUand iterate evaluation of the training examples over five to ten epochs,with one epoch taking one hour to complete. In other implementations,the variant classifier can be trained for 50 epochs, with smallimprovements after 20 to 30 epochs without overfitting

In some implementations, the target base position can be flanked by orpadded to at least 30 bases on each side.

The convolutional neural network can also have one or more max poolinglayers and one or more batch normalization layers.

In some implementations, the convolutional neural network can be trainedon one or more training servers. After the training, the convolutionalneural network can be deployed on one or more production servers(supporting a cloud environment) that receive the group of reads fromrequesting clients. The production servers can process the group ofreads through the input and output modules of the convolutional neuralnetwork to produce the classification scores that are transmitted to theclients.

Other implementations may include a non-transitory computer readablestorage medium storing instructions executable by a processor to performfunctions of the system described above.

In another implementation, the technology disclosed relates to a methodof variant calling. The method includes feeding an array of inputfeatures to a convolutional neural network and processing the arraythrough the convolutional neural network.

The array encodes a group of reads that are aligned to a reference readand include a target base position flanked by or padded to at least 30bases on each side. Each input feature in the array corresponds to abase in the reads and has a plurality of dimensions.

The plurality of dimensions includes a first dimension set identifyingthe base, a second dimension set identifying a reference base aligned tothe base, a third dimension set identifying a base call accuracy scoreof the base, a fourth dimension set identifying strandedness (e.g., DNAstrandedness) of the base, a fifth dimension set identifying aninsertion count of changes adjoining a position of the base, and a sixthdimension set identifying a deletion flag at the position of the base.

The method further includes translating processing of the array by theconvolutional neural network into classification scores for likelihoodthat each input feature at the target base position is a true variant ora false variant.

In some implementations, each input feature can have twelve dimensions.In some implementations, the first dimension set can encode four basesusing one-hot encoding. In some implementations, the second dimensionset can encode four bases using one-hot encoding.

Each of the features discussed in this particular implementation sectionfor the system implementations apply equally to this methodimplementation. As indicated above, all the system features are notrepeated here and should be considered repeated by reference.

Other implementations may include a non-transitory computer readablestorage medium storing instructions executable by a processor to performthe method described above. Yet another implementation may include asystem including memory and one or more processors operable to executeinstructions, stored in the memory, to perform the method describedabove.

In another implementation, the technology disclosed relates to a systemcomprising a trained variant classifier. The variant classifier includesnumerous processors operating in parallel and coupled to memory. Thevariant classifier also includes a convolutional neural network thatruns on the numerous processors.

The convolutional neural network is trained on at least 50000 to 1000000training examples of groups of reads spanning candidate variant siteslabeled with true variant classifications of the groups using abackpropagation-based gradient update technique that progressivelymatches outputs of the convolutional neural network with correspondingground truth labels.

Each of the training examples used in the training includes a group ofreads that are aligned to a reference read. Each of the reads includes atarget base position that is flanked by or padded to at least 110 baseson each side.

Each of the bases in the reads is accompanied by a correspondingreference base in the reference read, a base call accuracy score ofreading the base, a strandedness (i.e., DNA strandedness) of reading thebase, insertion count of changes adjoining a position of the base, anddeletion flag at the position of the base.

An input module of the convolutional neural network, which runs on atleast one of the numerous processors, feeds the group of reads forevaluation of the target base position.

An output module of the convolutional neural network, which runs on atleast one of the numerous processors, translates analysis by theconvolutional neural network into classification scores for likelihoodthat each candidate variant at the target base position is a truevariant or a false variant.

This system implementation and other systems disclosed optionallyinclude one or more of the following features. System can also includefeatures described in connection with methods disclosed. In the interestof conciseness, alternative combinations of system features are notindividually enumerated. Features applicable to systems, methods, andarticles of manufacture are not repeated for each statutory class set ofbase features. The reader will understand how features identified inthis section can readily be combined with base features in otherstatutory classes.

Each of the bases in the reads can be further accompanied by a mappingquality score of aligning a corresponding read that contains the base tothe reference read.

The convolutional neural network can have one or more convolution layersand one or more fully-connected layers. The convolutional neural networkcan process the group of reads through the convolution layers andconcatenate output of the convolution layers with correspondingempirical variant score (abbreviated EVS) features, and feed the resultof the concatenation to the fully-connected layers.

Each convolution layer has convolution filters and each of theconvolution filters has convolution kernels. The convolution filters canuse depthwise separable convolutions.

The convolutional neural network can have one or more max pooling layersand one or more batch normalization layers.

The convolutional neural network can use a softmax classification layerto produce the classification scores.

The convolutional neural network can use dropout.

The convolutional neural network can use flattening layers.

The convolutional neural network can use concatenation layers.

The convolutional neural network can run on a GPU and iterate evaluationof the training examples over five to fifty epochs, with one epochtaking one hour to complete.

Other implementations may include a non-transitory computer readablestorage medium storing instructions executable by a processor to performfunctions of the system described above.

In another implementation, the technology disclosed relates to a methodof variant calling. The method includes feeding an array of inputfeatures to a convolutional neural network and processing the arraythrough the convolutional neural network.

The convolutional neural network runs on numerous processors operatingin parallel and coupled to memory, and is trained on at least 50000training examples of groups of reads spanning candidate variant siteslabeled with true variant classifications of the groups using abackpropagation-based gradient update technique that progressivelymatches outputs of the convolutional neural network with correspondingground truth labels.

The array encodes a group of reads that are aligned to a reference readand include a target base position flanked by or padded to at least 30bases on each side. Each input feature in the array corresponds to abase in the reads and has a plurality of dimensions.

The plurality of dimensions includes a first dimension set identifyingthe base, a second dimension set identifying a reference base aligned tothe base, a third dimension set identifying a base call accuracy scoreof the base, a fourth dimension set identifying strandedness (e.g., DNAstrandedness) of the base, a fifth dimension set identifying aninsertion count of changes adjoining a position of the base, and a sixthdimension set identifying a deletion flag at the position of the base.

The method further includes translating processing of the array by theconvolutional neural network into classification scores for likelihoodthat each input feature at the target base position is a true variant ora false variant.

Each of the features discussed in this particular implementation sectionfor the system implementations apply equally to this methodimplementation. As indicated above, all the system features are notrepeated here and should be considered repeated by reference.

Other implementations may include a non-transitory computer readablestorage medium storing instructions executable by a processor to performthe method described above. Yet another implementation may include asystem including memory and one or more processors operable to executeinstructions, stored in the memory, to perform the method describedabove.

Fully-Connected Network (FCN) Implementations

In yet another implementation, the technology disclosed relates to asystem comprising a trained variant classifier. The variant classifierincludes numerous processors operating in parallel and coupled tomemory. The variant classifier also includes a fully-connected neuralnetwork that runs on the numerous processors.

The fully-connected neural network is trained on at least 50000 to1000000 training examples of empirical variant score (abbreviated EVS)feature sets of candidate variant sites labeled with true variantclassifications of the site using a backpropagation-based gradientupdate technique that progressively matches outputs of thefully-connected neural network with corresponding ground truth labels.

Each of the training examples used in the training includes an EVSfeature set representing characteristics of a corresponding candidatevariant site in a group of reads.

An input module of the fully-connected neural network, which runs on atleast one of the numerous processors, feeds the EVS feature set forevaluation of a target candidate variant site.

An output module of the fully-connected neural network, which runs on atleast one of the numerous processors, translates analysis by thefully-connected neural network into classification scores for likelihoodthat at least one variant occurring at the target candidate variant siteis a true variant or a false variant.

This system implementation and other systems disclosed optionallyinclude one or more of the following features. System can also includefeatures described in connection with methods disclosed. In the interestof conciseness, alternative combinations of system features are notindividually enumerated. Features applicable to systems, methods, andarticles of manufacture are not repeated for each statutory class set ofbase features. The reader will understand how features identified inthis section can readily be combined with base features in otherstatutory classes.

The fully-connected neural network can have one or more max poolinglayers and one or more batch normalization layers.

The fully-connected neural network can use dropout.

The fully-connected neural network can use a softmax classificationlayer to produce the classification scores.

Other implementations may include a non-transitory computer readablestorage medium storing instructions executable by a processor to performfunctions of the system described above.

In another implementation, the technology disclosed relates to a methodof variant calling. The method includes feeding an empirical variantscore (abbreviated EVS) feature set of a target candidate variant siteto a fully-connected neural network and processing the EVS feature setthrough the fully-connected neural network.

The fully-connected neural network runs on numerous processors operatingin parallel and coupled to memory, and is trained on at least 50000training examples of EVS feature sets of candidate variant sites labeledwith true variant classifications of the site using abackpropagation-based gradient update technique that progressivelymatches outputs of the fully-connected neural network with correspondingground truth labels.

The EVS feature set represents characteristics of the target candidatevariant site.

The method further includes translating processing of the EVS featureset by the fully-connected neural network into classification scores forlikelihood that at least one variant occurring at the target candidatevariant site is a true variant or a false variant.

Each of the features discussed in this particular implementation sectionfor the system implementations apply equally to this methodimplementation. As indicated above, all the system features are notrepeated here and should be considered repeated by reference.

Other implementations may include a non-transitory computer readablestorage medium storing instructions executable by a processor to performthe method described above. Yet another implementation may include asystem including memory and one or more processors operable to executeinstructions, stored in the memory, to perform the method describedabove.

The preceding description is presented to enable the making and use ofthe technology disclosed. Various modifications to the disclosedimplementations will be apparent, and the general principles definedherein may be applied to other implementations and applications withoutdeparting from the spirit and scope of the technology disclosed. Thus,the technology disclosed is not intended to be limited to theimplementations shown, but is to be accorded the widest scope consistentwith the principles and features disclosed herein. The scope of thetechnology disclosed is defined by the appended claims.

What is claimed is: 1-20. (canceled)
 21. A system comprising: at leastone processor; and a non-transitory computer readable medium storing aconvolutional neural network and instructions that, when executed by theat least one processor, cause the system to: identify a group of readsaligned with a reference genome and spanning a candidate variant at atarget base position; provide, to the convolutional neural network, anarray of input features encoding bases from the group of reads at thetarget base position, bases flanking each side of the target baseposition, and corresponding base features for bases within the group ofreads; and generate, based on an analysis of the array of input featuresby the convolutional neural network, classification scores indicatinglikelihoods that the candidate variant at the target base position is avariant.
 22. The system of claim 21, further comprising instructionsthat, when executed by the at least one processor, cause the system togenerate the classification scores by generating a first probabilitythat the candidate variant is a true variant and a second probabilitythat the candidate variant is a false variant.
 23. The system of claim21, further comprising instructions that, when executed by the at leastone processor, cause the system to generate the classification scores bygenerating one or more of a first score that the candidate variant is ahomozygous variant, a second score that the candidate variant is aheterozygous variant, a third score that the candidate variant is anon-variant, or a fourth score that the candidate variant is acomplex-variant.
 24. The system of claim 21, further comprisinginstructions that, when executed by the at least one processor, causethe system to: process the array of input features through one or morelayers of the convolutional neural network to generate convolutionaloutput features; concatenate the convolutional output features withper-variant characterization data corresponding to the candidate variantat the target base position to generate concatenated features; andgenerate the classification scores based on an analysis of theconcatenated features by one or more layers of the convolutional neuralnetwork.
 25. The system of claim 24, wherein the per-variantcharacterization data corresponding to the candidate variant compriseempirical variant score (EVS) features for the candidate variant. 26.The system of claim 24, further comprising instructions that, whenexecuted by the at least one processor, cause the system to generate theclassification scores based on the analysis of the concatenated featuresby one or more fully connected layers and a classification layer of theconvolutional neural network.
 27. The system of claim 24, furthercomprising instructions that, when executed by the at least oneprocessor, cause the system to process the array of input featuresthrough the one or more layers of the convolutional neural network byprocessing the array of input features through a one or more of an inputlayer, a convolution layer, a batch normalization layer, a max poolinglayer, or a flattening layer.
 28. The system of claim 21, furthercomprising instructions that, when executed by the at least oneprocessor, cause the system to provide the array of input features inwhich each feature corresponds to a base in a read from the group ofreads and each feature comprises a plurality of dimensions.
 29. Thesystem of claim 21, wherein the corresponding base features for baseswithin the group of reads comprise one or more of: a correspondingreference base in a reference read corresponding to the referencegenome; a base call accuracy score of calling a base in a read of thegroup of reads; a strandedness of calling the base; an insertion countof changes adjoining a position of the base; a deletion flag at theposition of the base; or a mapping quality score of aligning acorresponding read that contains the base to the reference read.
 30. Thesystem of claim 21, wherein the bases flanking each side of the targetbase position comprise one or more null bases representing positions atwhich reads from the group of reads lack corresponding base calls.
 31. Anon-transitory computer readable storage medium storing instructionsthat, when executed by at least one processor, cause a computing systemto: identify a group of reads aligned with a reference genome andspanning a candidate variant at a target base position; provide, to aconvolutional neural network, an array of input features encoding basesfrom the group of reads at the target base position, bases flanking eachside of the target base position, and corresponding base features forbases within the group of reads; and generate, based on an analysis ofthe array of input features by the convolutional neural network,classification scores indicating likelihoods that the candidate variantat the target base position is a variant.
 32. The non-transitorycomputer readable storage medium of claim 31, further storinginstructions that, when executed by the at least one processor, causethe computing system to generate the classification scores by generatinga first probability that the candidate variant is a true variant and asecond probability that the candidate variant is a false variant. 33.The non-transitory computer readable storage medium of claim 31, furtherstoring instructions that, when executed by the at least one processor,cause the computing system to generate the classification scores bygenerating one or more of a first score that the candidate variant is ahomozygous variant, a second score that the candidate variant is aheterozygous variant, a third score that the candidate variant is anon-variant, or a fourth score that the candidate variant is acomplex-variant.
 34. The non-transitory computer readable storage mediumof claim 31, further storing instructions that, when executed by the atleast one processor, cause the computing system to: process the array ofinput features through one or more layers of the convolutional neuralnetwork to generate convolutional output features; concatenate theconvolutional output features with per-variant characterization datacorresponding to the candidate variant at the target base position togenerate concatenated features; and generate the classification scoresbased on an analysis of the concatenated features by one or more layersof the convolutional neural network.
 35. The non-transitory computerreadable storage medium of claim 34, wherein the per-variantcharacterization data corresponding to the candidate variant compriseempirical variant score (EVS) features for the candidate variant. 36.The non-transitory computer readable storage medium of claim 34, furtherstoring instructions that, when executed by the at least one processor,cause the computing system to generate the classification scores basedon the analysis of the concatenated features by one or more fullyconnected layers and a classification layer of the convolutional neuralnetwork.
 37. A computer-implemented method comprising: identifying agroup of reads aligned with a reference genome and spanning a candidatevariant at a target base position; providing, to a convolutional neuralnetwork, an array of input features encoding bases from the group ofreads at the target base position, bases flanking each side of thetarget base position, and corresponding base features for bases withinthe group of reads; and generating, based on an analysis of the array ofinput features by the convolutional neural network, classificationscores indicating likelihoods that the candidate variant at the targetbase position is a variant.
 38. The computer-implemented method of claim37, wherein generating the classification scores comprises generating afirst probability that the candidate variant is a true variant and asecond probability that the candidate variant is a false variant. 39.The computer-implemented method of claim 37, wherein generating theclassification scores comprises generating one or more of a first scorethat the candidate variant is a homozygous variant, a second score thatthe candidate variant is a heterozygous variant, a third score that thecandidate variant is a non-variant, or a fourth score that the candidatevariant is a complex-variant.
 40. The computer-implemented method ofclaim 37, wherein the corresponding base features for bases within thegroup of reads comprise one or more of: a corresponding reference basein a reference read corresponding to the reference genome; a base callaccuracy score of calling a base in a read of the group of reads; astrandedness of calling the base; an insertion count of changesadjoining a position of the base; a deletion flag at the position of thebase; or a mapping quality score of aligning a corresponding read thatcontains the base to the reference read.